INVESTIGATION OF THE LIPOPROTEOME OF THE LYME DISEASE BACTERIUM

BORRELIA BURGDORFERI

BY

Alexander S. Dowdell

Submitted to the graduate degree program in Microbiology, Molecular Genetics & Immunology

and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements

for the degree of Doctor of Philosophy.

_____________________________

Wolfram R. Zückert, Ph.D., Chairperson

_____________________________

Indranil Biswas, Ph.D.

_____________________________

Mark Fisher, Ph.D.

_____________________________

Joe Lutkenhaus, Ph.D.

_____________________________

Michael Parmely, Ph.D.

Date Defended: April 27th, 2017

ii

The dissertation committee for Alexander S. Dowdell certifies that this is the approved version

of the following dissertation:

INVESTIGATION OF THE LIPOPROTEOME OF THE LYME DISEASE BACTERIUM

BORRELIA BURGDORFERI

_____________________________

Wolfram R. Zückert, Ph.D., Chairperson

Date Approved: May 4th, 2017

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Abstract

The spirochete bacterium Borrelia burgdorferi is the causative agent of Lyme borreliosis, the top

vector-borne disease in the United States. B. burgdorferi is transmitted by hard-

bodied Ixodes ticks in an enzootic tick/vertebrate cycle, with human infection occurring in an

accidental, “dead-end” fashion. Despite the estimated 300,000 cases that occur each year, no

FDA-approved vaccine is available for the prevention of Lyme borreliosis in humans.

Development of new prophylaxes is constrained by the limited understanding of the

pathobiology of B. burgdorferi, as past investigations have focused intensely on just a handful of

identified proteins that play key roles in the tick/vertebrate infection cycle. As such,

identification of novel B. burgdorferi virulence factors is needed in order to expedite the

discovery of new anti-Lyme therapeutics. The multitude of lipoproteins expressed by the

spirochete fall into one such category of virulence factor that merits further study. These

lipoproteins play diverse roles in the organism and localize to different membrane-peripheral

cellular compartments.

The overarching goal of the current study was to further define the structure-function of the B.

burgdorferi cell envelope by comprehensively and conclusively defining the localization of the

bacterium’s predicted lipoproteome, using an epitope-tagged lipoprotein expression library. The

data show that the majority of B. burgdorferi lipoproteins are surface-exposed, and that the

plasmids of B. burgdorferi are enriched in surface lipoprotein genes relative to the chromosome.

The study also establishes and validates a high-throughput proteomics approach that can be used

in future studies to assess localization of endogenously expressed untagged lipoproteins. Finally,

an analysis of the lipoproteome using data mining and in silico fold recognition algorithms

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demonstrates potential roles for several uncharacterized lipoproteins and identifies new targets

for vaccine development.

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Acknowledgements

I’d like to thank Dr. Wolfram Zückert for providing me with funding, support, and advice over

the past several years. I couldn’t have asked for a better graduate experience or mentor. I’d

also like to thank my graduate committee and the other faculty of University of Kansas Medical

Center for providing helpful advice and for always pushing me to become a better scientist. I’d

like to thank the administrative staff of the Department of Microbiology, Molecular Genetics &

Immunology for always being willing to help me throughout my graduate career. Finally, I’d

like to thank my family and friends for all of the support throughout my Ph.D. studies. I’d

especially like to thank my amazing wife Anita for sticking with me throughout all of the

working weekends and late nights at the lab. I’m finally done, sweetest heart!

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Table of Contents Chapter 1: Background.................................................................................................................1 Chapter 2: Lipoprotein Biosynthesis in B. burgdorferi............................................................20 Chapter 3: Previous Research of B. burgdorferi Lipoproteins................................................38 Chapter 4: Localization of B. burgdorferi Lipoproteome.........................................................41 Chapter 5: Detailed Experimental Protocols............................................................................89 Chapter 6: In-depth Analysis of Selected B. burgdorferi Lipoproteins................................104 Appendix: Supplemental Figures and Tables.........................................................................123 References...................................................................................................................................140

Table of Figures and Tables Figure 1. Sec-dependent translocation of lipoproteins............................................................23 Figure 2. Post-translational modification of lipoproteins.......................................................27 Figure 3. The sorting of lipoproteins via Lol machinery.........................................................31 Figure 4. Alignment of the B. burgdorferi OppA homolog tether sequences.........................35 Figure 5. Plasmid map of the expression vector pSC-LP........................................................43 Figure 6. Surface proteolysis of B. burgdorferi strains using Proteinase K...........................63 Figure 7. Membrane fractionation of B. burgdorferi...............................................................69 Figure 8. Surface proteolysis of B. burgdorferi using Pronase................................................73 Figure 9. Membrane fractionation of B. burgdorferi...............................................................77 Figure 10. Sequence alignment of B. burgdorferi tether peptides...........................................81 Figure 11. Structural Analysis of BB0155 using I-TASSER.................................................113 Figure 12. COACH Analysis of BB0352.................................................................................117 Figure S1. Statistical analyses of quantitative proteomics features.....................................123 Figure S2. Graphical Representation of putative lipoproteins..............................................125

Table 1. List of oligonucleotide primers used for lipoprotein amplification.........................47 Table 2. B. burgdorferi lipoproteome localization data..........................................................55 Table 3. Comparison of lipoprotein tether length based on localization...............................85 Table 4. Analysis of Lipoprotein Genes by HMMSCAN......................................................105 Table 5. Analysis of putative TPR Lipoproteins....................................................................107 Table 6. Remote Homology Analysis of B. burgdorferi Lipoproteins..................................109 Table S1. Detailed peptide and spectral counts of MudPIT-detected proteins.....................85

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Chapter 1: Background

1.1 History of Lyme borreliosis (Lyme Disease)

Although Lyme borreliosis has only recently been described and the etiologic agent

determined to be the spirochete Borrelia burgdorferi, evidence exists that humanity has been

afflicted with this disease for much longer. European researchers had long known that the bite of

certain hard-bodied ticks could results in a disease with dermatological and neurological

complications (1). One of the first documented presentations of possible Lyme borreliosis came

from Arvid Afzelius, a founder of the Swedish Society of Dermatology, in 1909. He presented

the case of an elderly woman with the now-characteristic expanding annular erythema, which he

termed “erythema migrans”, at the site of an Ixodes reduvius tick bite. Subsequent clinical

observations of tick-bitten patients found similar dermatological manifestations and occasionally

observed pathology of the central nervous system (lymphocytic meningitis). The 1922 findings

of Garin & Bujadoux are believed to be the first recorded description of Lyme neuroborreliosis,

although a retrospective diagnosis of their patient has led to speculation that he may have been

co-infected with other tick-borne pathogens (2, 3). Researchers in Europe eventually concluded

that a causal link existed between the disease and ticks, with speculation that a tick salivary

gland toxin or tick-associated pathogen was ultimately responsible (4). Evidence exists that

human infection by B. burgdorferi may even predate these published accounts. PCR analysis of

DNA present in the Tyrolean Iceman “Ötzi”, a 5,000-year-old preserved mummy from the

Italian Alps, showed that he had been infected with B. burgdorferi and is the oldest known

human to have had Lyme borreliosis (5).

In 1975, the Connecticut State Health Department was contacted concerning an epidemic

of juvenile rheumatoid arthritis in Lyme, Connecticut, with an unknown etiology. Dr. Allen

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Steere, working on behalf of a joint investigation between the Yale University School of

Medicine and Connecticut State Health Department, published his observations of oligoarticular

arthritis in both children and adults (6). Investigation showed that juvenile rheumatic arthritis

was not the cause of the observed symptoms, as the incidence of reported arthritis was 100 times

the expected prevalence, and consequently the new affliction was termed “Lyme arthritis”.

Many patients also developed expanding erythematous lesions reminiscent of those observed in

Europe, and a further subset of patients also developed cardiological and/or neurological

complications (7). Although the causative agent of Lyme arthritis was still known, it was

demonstrated that early administration of antibiotics could ameliorate the symptoms of the

disease (8). Epidemiological studies implicated Ixodes ticks with the transmission of disease,

similar to the previous findings in Europe (9).

The identification of the spirochete B. burgdorferi came later, during 1978 and 1979

investigations into outbreaks of the tickborne Apicomplexan parasite babesiosis in the Northeast.

Serosurveys conducted in Shelter Island, New York, identified a number of individuals who did

not have babesiosis but instead reported symptoms resembling patients from Lyme, Connecticut.

Sera from these patients were reactive to spirochetes grown in Kelly’s Medium from ticks

collected on Shelter Island, demonstrating a connection between these spirochetes and the

disease from Lyme (10). These spirochetes were also later isolated directly from patients with

Lyme disease, further strengthening the evidence that this bacterium was the causative agent (11,

12). The organism was named Borrelia burgdorferi to honor Dr. Willy Burgdorfer’s efforts in

the organism’s discovery, with its accompanying disease renamed “Lyme borreliosis” (13).

Today, Lyme borreliosis is the most common vector-borne disease in the United States, and an

estimated 300,000 cases occur each year (14). There is no FDA-approved vaccine currently

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available for the prevention of Lyme borreliosis. Lyme borreliosis is considered an emerging

disease and research into the pathogenesis of B. burgdorferi, as well as prophylaxis and

treatments, is ongoing (15).

1.2 The Enzootic Cycle of B. burgdorferi

In nature, B. burgdorferi exists in a complex relationship with its tick and vertebrate

hosts, transitioning between the two at different stages of the tick life cycle. Only certain hard-

bodied ticks of the genus Ixodes can become infected with B. burgdorferi, and the molecular

specificity of the tick-spirochete interaction suggests that B. burgdorferi has evolved to utilize

this genus of tick as the means by which it spreads between vertebrates. B. burgdorferi is not

transmitted transovarially in ticks, and newly hatched larvae acquire the bacterium from a

previously infected host (16, 17). The transmission cycle typically begins with an uninfected

tick larva feeding on an infected vertebrate host, typically a small bird or mammal. The white-

footed mouse, Peromyscus leucopus, is especially notable as an important natural reservoir of B.

burgdorferi in North America. Interestingly, laboratory infected adult Peromyscus mice can

become persistently infected with B. burgdorferi but do not develop many of the pathologies

associated with human infection, suggesting that host factors may modulate the severity of B.

burgdorferi infection (18). Spirochetes can be detected in the feeding tick within 12 hours from

the start of feeding, and bacterial colonization of the tick continues for the full extent of the

feeding period (typically 72-96 hours) (16, 17). B. burgdorferi takes up residence in the tick

midgut, wherein it can persist for several months while the larval tick molts into a nymph.

During this time, the tick does not feed and displays limited metabolic activity. The molecular

mechanisms by which B. burgdorferi senses host feeding and transits to the tick, as well as how

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it survives in the nutrient-depleted midgut for months, are unclear at this time. Microarray

studies examining the transcriptome of B. burgdorferi in larvae, nymphs, and dialysis membrane

chambers have shed light on which genes are important for certain stages of the tick/vertebrate

cycle; however, many of these genes encode proteins without identifiable functions or homologs

in non-spirochetes, thus preventing full understanding of the molecular mechanism behind the B.

burgdorferi tick/vertebrate life cycle (19).

After molting, the unfed nymph then proceeds to feed on a second host, usually of the

same variety chosen by the unfed larvae (16, 17). Transmission of B. burgdorferi to the

uninfected vertebrate host occurs at this stage and replenishes the B. burgdorferi reservoir in

small animals. Accidental transmission to humans, dogs, and other “accidental” hosts also occur

at this stage. The steps leading to transmission of B. burgdorferi have been well-characterized.

During the first 36 hours of tick feeding, B. burgdorferi in the tick midgut is immersed in

undigested blood from the host and begins a period of rapid cell division and dissemination. The

bacteria penetrate the tick gut and migrate to the salivary gland, wherein they pass into the

vertebrate host and begin further dissemination. Interestingly, this process of transmission can

take up to 72 hours for B. burgdorferi but can occur in less than an hour for relapsing fever

Borrelia spirochetes, which colonize fast-feeding soft-bodied ticks (20). Tick salivary gland

proteins are thought to play a beneficial role in B. burgdorferi transmission based on gene

expression and bacterial surface binding studies (21, 22). The fed nymphs then detach from their

hosts and, after several months, molt into male and female adult ticks. Adult ticks seem to play

little role in the transmission of B. burgdorferi to humans, although they play an important role

in nature through perpetuation of the tick population.

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1.3 The Fundamental Role of B. burgdorferi Lipoproteins

Lipid-modified proteins, or “lipoproteins”, are ubiquitous in both Gram-positive and

Gram-negative bacteria. B. burgdorferi is noteworthy in that it dedicates a large percentage of

its genome to the expression of lipoproteins, with one estimate placing the number of lipoprotein

encoding-genes at about 8% of all reading frames (23). This is especially significant considering

that the B. burgdorferi genome is highly reduced at approximately 1.314 MBp (24).

Lipoproteins are peripheral membrane proteins, anchored to either the inner or outer membrane

through a conserved, acylated cysteine residue at the N terminus (also see 2. Lipoprotein

Biosynthesis in B. burgdorferi) (25). Following the acylated cysteine is a span of disordered

residues (the “tether”) which, in Proteobacteria, contains the necessary information for

lipoprotein sorting at the inner membrane (26). Prelimiary evidence shows that, although this

region can modulate surface exposure of outer membrane lipoproteins, it does not seem to play a

role in the release of outer membrane lipoproteins at the inner membrane (27-30). In B.

burgdorferi, tether regions are highly variable in both length and amino acid content, with no

obvious correlation existing between a lipoprotein’s localization and its tether

length/biochemical properties (Table 3) (27). However, the length of surface lipoprotein tethers

may act to position the lipoprotein in a distinct spatial setting relative to other membrane

proteins, with its positioning possibly tied to the biological role of the lipoprotein (25).

Following the tether region is a more constrained region of the protein that is believed to be

responsible for the lipoprotein’s biological function.

The lipoproteins of B. burgdorferi play diverse roles in the organism, with identified

functions ranging from housekeeping duties to host interactions, and consequentially these

lipoproteins are necessary for both in vitro growth using artificial culture medium as well as the

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more complex tick/vertebrate transmission cycle found in nature. Many lipoproteins also have

no identifiable function or homology, yet are indispensable to B. burgdorferi at a specific stage

in its life cycle. Here, the various archetypes of lipoproteins will be discussed using well-

researched, prototypical examples.

Certain B. burgdorferi lipoprotein play important housekeeping functions to the organism

and are among those that show the most homology to proteins in other bacteria (31, 32). These

lipoproteins often act as nutrient uptake factors and are frequently found as part of inner

membrane ATP-binding cassette (ABC) transporters. These lipoproteins, called substrate-

binding proteins, are responsible for coordinating the binding and ATP hydrolysis-driven uptake

of specific solutes from the periplasm into the cytoplasm (33). Of these substrate-binding

lipoproteins, some of the most prominent belong to the Oligopeptide Permease (OppA) family of

substrate-binding proteins. These proteins facilitate the transport of small peptides from the

periplasm into the cytoplasm through interaction with the ABC transport subunits OppB/C/D/F.

B. burgdorferi, interestingly, expresses five copies of oppA: three on the linear chromosome, one

on the mini-chromosome cp26, and one on the dispensable linear plasmid lp54 (34, 35). These

copies seem to have arisen from paralogous gene duplication, as they are homologous in amino

acid sequence (≥44% pair-wise identity) but show different peptide binding affinities (34, 36).

Transposon mutagenesis screens suggest that these genes are not individually essential, implying

that there may be some overlap or redundancy in their substrate specificity that permits growth

of B. burgdorferi in vitro (37). The authors of this study also came to the conclusion that the

OppA homologs in B. burgdorferi could complement each other in vitro. The B. burgdorferi

gene oppA-1 (BB0328) was found to be able to complement an E. coli ΔoppA strain, further

supporting the conservation of these proteins between disparate bacterial lineages (38). The

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OppA proteins also show individual transcriptional regulation patterns based on environmental

cues and host species, indicating that their expression may correlate with different nutritional

demands at various phases of the tick/vertebrate cycle through their dissimilar peptide binding.

Further research of their transcription showed that oppA-1 (BB0328), oppA-2 (BB0329), and

oppA-3 (BB0330) can be transcribed tricistronically, bicistronically, or individually, and that

these genes were not regulated by temperature (34). In contrast, the gene oppAV (BBA34) is

under control of the alternative sigma factors RpoN (BB0450) and RpoS (BB0771), implicating

the expression of this gene in the vertebrate stage of infection and supporting previous findings

that this OppA gene is the only one regulated by temperature(34, 39, 40). Similarly, the gene

oppAIV (BBB16) showed regulation by the B. burgdorferi BosR/Fur homolog (BB0647), a

transcriptional regulator that has been associated with expression of virulence genes in

vertebrates (41). These data suggest that the genes oppAIV and oppAV may be upregulated

during transmission of B. burgdorferi from the tick to the vertebrate host. Despite this evidence

implicating oppAIV and oppAV in the infection cycle, transposon mutagenesis studies and gene

deletion experiments have not conclusively demonstrated that either gene is required for

infection in the mouse model (37, 42). In addition to OppA homologs, other substrate-binding

proteins have been identified in B. burgdorferi. The protein ProX (BB0144) is a predicted L-

proline/glycine betaine substrate-binding protein and is situated in a likely operon with putative

ABC transporter components (31, 32). Based on homology, it likely functions in transport of

osmoregulatory molecules into the cell; however, this has not been demonstrated experimentally.

The protein PstS (BB0215) is also predicted to be a substrate-binding protein, with homology to

characterized phosphate-binding proteins. Similar to ProX, PstS exists in a possible operon with

predicted ABC transporter proteins. Surprisingly, binding assays performed with soluble,

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recombinant PstS show that the protein binds to sulfate anions with at least 20-fold greater

affinity than for phosphate, suggesting that PstS may be involved in the transport of both anions

into the cytoplasm (43). Transposon mutagenesis assays suggest that both ProX and PstS are

essential for in vitro cultivation of B. burgdorferi, consistent with their predicted role in substrate

transport (37). One interesting observation of B. burgdorferi substrate-binding proteins is that

they appear to be lipoproteins, based on conserved cysteine residues and a putative lipobox motif

(23). The structure of these proteins in B. burgdorferi contrasts with that of substrate-binding

proteins in E. coli and other diderms, in which they are soluble periplasmic proteins rather than

peripheral, membrane anchored proteins (33). In this regard, the substrate-binding proteins of B.

burgdorferi are more similar to those of Gram-positive bacteria, which also express substrate-

binding proteins as lipoproteins. However, the presence of outer membrane biogenesis

machinery, such as the Bam and Lol complexes, suggests that the most recent common ancestor

of B. burgdorferi with non-spirochetes was Gram-negative, a conclusion supported by 16S rRNA

sequencing and alignment (44). Although the evolution of B. burgdorferi is unclear based on the

early divergence of the phylum Spirochaetes, it is most likely that convergent evolution is

responsible for the observation that the substrate-binding proteins of both B. burgdorferi and

Gram-positive organisms are lipoproteins.

In addition to such metabolic roles, certain B. burgdorferi lipoproteins are also mediators

of pathogenicity. These lipoproteins have been shown to be important in various stages of the

tick/vertebrate infectious cycle, with data showing that their loss interrupts the cycle at a point

that corresponds to the biological function of the protein. One such way that lipoproteins

mediate pathogenicity is through acting as surface adhesion proteins. Expression of such

lipoproteins facilitates survival in the tick vector and infection of the vertebrate host. OspA

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(BBA15), an abundant surface lipoprotein, was one of the first B. burgdorferi adhesins to be

discovered. OspA was first reported as a shared antigen on both human- and tick-isolated B.

burgdorferi based on affinity of a monoclonal antibody H5332, and it was later determined

through [3H]-palmitate metabolic labeling and radioimmunoprecipitation that OspA was

lipidated in B. burgdorferi (45, 46). Lipidated OspA was also found to be highly immunogenic

in mice, and inoculation with lipidated OspA could result in a protective immune response

against subsequent intradermal challenges with live B. burgdorferi (47). This study and others

were the foundation for the first FDA-approved anti-Lyme vaccine, LYMErix, which was based

on recombinant OspA as the immunogen (48). The discovery of OspA as an adhesin was

preceded by findings that OspA was selectively expressed in the tick vector and downregulated

upon transmission to the vertebrate host, leading to speculation that OspA plays a role in tick

colonization (49). Further work using tick gut extracts and yeast two-hybrid experiments

determined that OspA binds to a tick gut epithelium protein called TROSPA (Tick Receptor for

OspA) (50, 51). Studies using infectious B. burgdorferi isolates found that OspA is not required

for infection or persistence in the mouse model, but the loss of OspA prevents re-acquisition of

B. burgdorferi by uninfected feeding ticks (52). These results show that OspA acts to adhere B.

burgdorferi to the midgut epithelium during tick colonization and that it is essential for

completion of the natural tick/vertebrate infection cycle. OspA exists in a bicistronic operon

with OspB, a lipoprotein with similar amino acid sequence and structure to OspA (53-55). Tick

acquisition assays suggest that OspA and OspB are functionally and that spirochetes expressing

solely OspB can colonize and survive in larval ticks (56). Likewise, expression of OspA alone

has been shown to permit survival of B. burgdorferi in nymphal ticks after microinjection (52).

Expression of both OspA and OspB, though, has been shown to permit a greater extent of tick

10

colonization that expression of just OspA, suggesting that these two proteins play

complementary, but distinct, functions in vivo (52). The current working hypothesis is that

OspA and OspB arose from a gene duplication event and have diverged to some extent; however,

their precise biological function in relation to each other still has yet to be fully understood.

Besides OspA and OspB, other lipoproteins have been shown to act as B. burgdorferi adhesins in

the vertebrate host. These lipoproteins presumably facilitate the dissemination of B. burgdorferi

throughout various tissues. One prominent surface adhesin is the fibronectin binding protein P47

(FBP; BBK32). Fibronectin, a large mammalian glycoprotein, is a component of the

extracellular matrix and binds a variety of biological polymers such as heparin and collagen. B.

burgdorferi cell lysates were shown to bind fibronectin after transfer to nitrocellulose

membranes, resulting in a single unique band corresponding to FBP (57). Adhesion of B.

burgdorferi onto fibronectin-coated glass slides was also competitively inhibited by pre-

incubation with purified FBP, showing that fibronectin binding is solely due to FBP. Despite

these results, disruption of FBP was shown to have no effect on B. burgdorferi pathogenicity

(58). These results suggest that fibronectin binding is not essential to infectivity, likely due to

the actions of other B. burgdorferi adhesins. These other adhesins include the decorin-binding

proteins A and B (DbpA/B, BBA24/25). Decorin, a proteoglycan that associates closely with

collagen, was shown to facilitate the adhesion of B. burgdorferi to the wells of microtiter plates

and, through radiolabeling, was also shown to be specifically bound by the spirochetes (59). B.

burgdorferi that had their dbpAB genes disrupted were nevertheless able to fully complete the

tick/vertebrate infection cycle, suggesting that, like FBP, other surface adhesins may be able to

compensate for the lack of decorin binding in vivo (60). Finally, it has been established that the

putative lipoprotein ErpX (BBQ47) binds laminin, a family of large glycoproteins that are

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components of the mammalian extracellular matrix (61). ErpX belongs to a family of

paralogous, plasmid-encoded lipoproteins (Erp) that are up-regulated during transmission to the

vertebrate host and are immunogenic (62). As the authors demonstrated, B. burgdorferi

selectively bound to laminin-coated slides and that this binding could be diminished through

addition of soluble, recombinant ErpX. Although the role of ErpX in the infection cycle of B.

burgdorferi has not been investigated, it is highly probable that it, like the other B. burgdorferi

adhesins, is singularly dispensable for infection in the mouse model due to compensation effects.

Other lipoproteins are associated with transmission of B. burgdorferi and the

establishment in the vertebrate host. These lipoproteins play a critical role in the natural

tick/vertebrate enzootic cycle of B. burgdorferi, and their absence results in a noticeable

impediment to infection. The surface lipoprotein OspC is the prototypical example of such a

protein. It is selectively up-regulated by an increase in temperature or pH, that is, a shift to

conditions that mimic those of the vertebrate host (49, 63). Examination of the temporal

regulation of OspC in vivo has revealed that it is rapidly induced during tick feeding, with

immunofluorescence assays showing that 75% of spirochetes expressed the lipoprotein after 48

hours of tick feeding (64). Interestingly, the expression of OspA appeared to be somewhat

reciprocal to that of OspC, in which OspA-expressing cells decreased to 40% of the total

population by 96 hours (49, 64). The increase in temperature and pH flux appears to be the

driving factor of OspC expression, as spriochetes that are continually maintained at the

temperature and pH of a vertebrate host slowly down-regulate expression of OspC (64). OspC

has been shown to bind the tick salivary gland protein Salp15, an immunomodulatory factor that

is believed to allow B. burgdorferi to establish itself in the host. Salp15 has been shown to

inhibit the activation of CD4+ T-cells through binding of the CD4 receptor and has also been

12

demonstrated to modulate the action of dendritic cells through binding to the C-type lectin DC-

SIGN, ultimately resulting in lower dendritic cell activation and lower levels of cytokine release

(65-67). Salp15 has also been implicated in resistance to serum complement through inhibiting

the deposition of membrane attack complex factors (as have other lipoproteins, see next section)

(68). In addition to Salp15, OspC has been shown to the bind serum plasminogen, which is

subsequently converted to enzymatically active plasmin (69). This plasmin, still bound to the

surface of B. burgdorferi, has been demonstrated to enhance penetration of the endothelium by

B. burgdorferi and allow it to disseminate beyond the site of tick feeding (70). Corresponding to

these identified biological roles, inactivation of OspC in B. burgdorferi abrogated infectivity in

the mouse model either through needle inoculation or through tick feeding, demonstrating the

necessity of this lipoprotein for the transmission of B. burgdorferi (71).

In order for B. burgdorferi to establish a persistent infection in the vertebrate host, it must

tirelessly evade the host’s immune defenses. These defenses compose a variety of distinct anti-

bacterial responses that interact with specialized B. burgdorferi immune evasion proteins. One

way that B. burgdorferi evades the immune response so is by avoiding destruction by the host’s

complement system. The surface lipoprotein CRASP-1 (CspA; BBA68) is noteworthy in this

regard as it binds the complement regulatory proteins Factor H and Factor H-like protein 1

(FHL-1), soluble plasma proteins that are implicated in endogenous regulation of the alternative

complement pathway(72, 73). Factor H and FHL-1 bound in this way were found to be

biologically active, and expression of CRASP-1 correlated with resistance to serum complement

in vitro (73, 74). CRASP-1 was also found to be upregulated during infection of the vertebrate

host and downregulated during tick acquisition, further supporting the role of this lipoprotein in

host complement evasion (75). In addition to CRASP-1, B. burgdorferi has been shown to

13

expresses one additional Factor H/FHL-1 binding protein CRASP-2 (CspZ; BBH06) and three

additional Factor H binding proteins CRASP-3 (ErpP; BBN38), CRASP-4 (ErpC, no locus tag

available), and CRASP-5 (ErpA; BBL39) (76). In addition to binding Factor H, CRASP-3 and

CRASP-5 have been shown to bind CFHR-2 and CFHR-5, two additional complement

regulatory proteins (77). A recent study also found that recombinant, soluble CRASP-1 and

CRASP-3, but not other CRASPs, could inhibit hemolysis of rabbit erythrocytes by the

alternative complement pathway (78). CRASP-1 was further shown to inhibit all three

complement pathways through binding of the terminal complement factors C7 and C9, thereby

directly preventing assembly of the membrane attack complex and avoiding complement-

mediated lysis. These functional differences are possibly one reason why CRASP-2-deficient B.

burgdorferi remained resistant to complement and retained full infectivity in mice (79).

Interestingly, B. burgdorferi could still infect mice deficient in Factor H, further supporting the

hypothesis that certain CRASPs may bind complement regulatory proteins somewhat

promiscuously (80). Taken together, these results suggest that the interplay between CRASP

proteins and the complement system is complex and dynamic, and it can be surmised that these

lipoproteins evolved to have complementary, but distinct, functions in preventing complement-

mediated lysis of B. burgdorferi.

Besides neutralization of the host’s complement system, B. burgdorferi is also able to

evade the humoral immune response so that it can persistently infect its host. Although various

anti-B. burgdorferi antibodies can be detected in Lyme borreliosis patients, the bacterium is

nevertheless able to survive in the face of a targeted humoral response. One important way that

B. burgdorferi evades the host’s humoral defense is through expression of the surface lipoprotein

Vmp-like sequence E (VlsE; BBF0041). VlsE undergoes homologous recombination in vivo

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with 15 neighboring, silent vls cassettes that results in antigenic variation of the lipoprotein (81).

Lipidation of VlsE was confirmed through [3H]-palmitate metabolic labeling of B. burgdorferi

and radioimmunoprecipitation. This antigenic variation allows for evasion of previously

generated anti-VlsE antibodies, keeping B. burgdorferi one step ahead of the host and forcing

adaptation of the antibody response against the new VlsE antigen. Deletion studies of the vls

genetic locus showed that not only is expression of the VlsE lipoprotein necessary for persistence

of infection in the mouse model, but recombination of VlsE with the silent vls cassettes is

indispensable as well (82). It was also observed that recombination of VlsE occurred

continuously throughout the infection of mice by B. burgdorferi, and the study’s authors

hypothesized that VlsE recombination does not cease while B. burgdorferi persists in the

vertebrate host and would result in an immense number of VlsE serotypes (83). A subsequent

study found that VlsE does not undergo recombination in the tick vector, conclusively

demonstrating that VlsE recombination is a vertebrate-specific phenomenon (84). The

importance of VlsE antigenic variation was further shown in experiments conducted with severe

combined immunodeficient (SCID) mice, which lack an adaptive immune response, versus

immunocompetent controls (85). B. burgdorferi recovered from immunocompetent mice

showed VlsE recombination at a higher frequency and with a greater amount of complexity as

compared with spirochetes from SCID mice, suggesting that the adaptive immune response

places selection pressure directly on VlsE to maintain an antigenically novel façade. These

results, in total, show that the lipoprotein VlsE is a novel means by which B. burgdorferi evades

the host immune system in order to establish a persistent infection.

Finally, certain uncharacterized proteins are important for the tick/vertebrate enzootic

cycle. These proteins have been shown to be crucial for certain steps of infection, yet lack

15

characterization of their molecular function and have no identifiable non-spirochete homologs.

One such lipoprotein is the small, abundant protein Lp6.6 (BBA62). Lp6.6 was first identified as

mitogenic, low molecular weight protein with pronounced abundancy in B. burgdorferi,

accounting for about 2% of the cell dry weight (86). [3H]-palmitate labeling of B. burgdorferi

and subsequent radioimmunoprecipitation with the monoclonal anti-Lp6.6 antibody 270.7

demonstrated that Lp6.6 is indeed a lipoprotein. Localization assays revealed that Lp6.6

localized to the inner leaflet of the outer membrane, and is one of only a few lipoproteins to

localize in such a way (27, 86, 87). Lp6.6 was found to be up-regulated during tick acquisition

and down-regulated during transmission to the vertebrate host (88). Interestingly, B. burgdorferi

cells lacking Lp6.6 retained full infectivity in mice by needle inoculation, and uninfected ticks

that fed on these mice acquired the bacteria equally to those expressing Lp6.6. However, the

absence of Lp6.6 led to reduced spirochete load in mouse tissues when transmission occurred by

tick feeding, and qRT-PCR analysis of tick tissues showed that bacterial populations in the gut

and salivary glands were reduced 5- to 10-fold as compared with wild-type cells. It was also

found that Lp6.6 exists in outer membrane complexes with other pathogenesis-associated

lipoproteins, such as OspA (88, 89). Structural analysis of Lp6.6 showed that it exists primarily

in aggregation-prone α-helices and disordered stretches, and it is hypothesized that its

unconstrained tertiary structure may be important for its biological role (90). Another similarly

uncharacterized factor is the lipoprotein (Ip)LA7 (P22; BB0365). LA7 was first identified based

on reactivity with Lyme patient sera: it appeared as an approximately 22 kilodalton protein with

an acidic pI (5.7) that was chromosomally encoded and lipidated, based on [3H]-palmitate assays

(91, 92). Further studies determined that it localized primarily to the inner membrane of the

periplasm and that its expression could be induced by a temperature shift, pH shift, or through

16

addition of the autoinducer-2 precursor 4,5-dihydroxy-2,3-pentanedione (27, 93). This

regulation may be due to a putative binding site 5’ of la7 for the DNA-binding protein EbfC

(BB0462), which has been implicated in the vertebrate host-induced expression of other plasmid-

encoded lipoproteins (94, 95). Correspondingly, expression of LA7 in tick midguts declined

significantly as compared with expression in the mouse model, suggesting that LA7 is

specifically expressed in the vertebrate host (93). Recent studies also found that LA7 expression

spiked during tick feeding, and deficiency of LA7 in B. burgdorferi resulted in pronounced

inability to both transmit to mice and re-colonize uninfected ticks (96, 97). LA7-deficient

spirochetes were also significantly impaired in their ability to migrate to tick salivary glands

following the initiation of feeding. The combined results of LA7 expression and inactivation

data suggest that LA7 may play a role in chemotaxis of the organism, allowing it to recognize the

need to disseminate to a new host. The absence of LA7, in turn, would impair the spirochete’s

recognition of chemotaxis or dissemination signals. This hypothesis is supported by data

showing that inactivation of LA7 does not impair B. burgdorferi’s ability to maintain itself

within the tick or to establish infection in the mouse after needle inoculation (96, 97). Further

support is found in that inactivation of a B. burgdorferi luxS homolog also does not interfere with

infection of mice by needle inoculation, which then suggests that chemotaxis proteins in general

may not be essential for establishing a persistent infection in vertebrates (98).

1.4 Development of Anti-Lyme borreliosis Vaccines

There is currently no FDA-approved vaccine to prevent Lyme borreliosis, despite the 300,000+

cases of the disease that occur each year (14). The lack of preventative measures against B.

burgdorferi infection has numerous implications, the economic burden of which has been

17

estimated at over $16,000 for a single late-stage/chronic Lyme patient (14). A vaccine would be

highly beneficial to areas where B. burgdorferi is endemic, as 95% of 2015 Lyme borreliosis

cases occurred in just two geographic foci comprising 14 U.S. states (14). Past research has

demonstrated that numerous B. burgdorferi proteins are naturally immunogenic; however, the

majority of natural B. burgdorferi immunogens occur on the spirochete’s plasmids, which are

naturally less stable and more dynamic than the chromosome (99, 100). However, it has also

been observed that the plasmids cp26 and lp54 are highly conserved in all clinical B. burgdorferi

isolates, and therefore it is no surprise that the immunogenic proteins from these genetic

elements were among the first to be examined for potential as vaccine candidates (101, 102).

OspA, as noted in the previous section, plays an indispensable role in the tick/vertebrate

transmission cycle and has been thoroughly investigated for potential as a vaccinogen. It has

been observed that pre- or co-administration of an OspA-based antibody to mice inhibited the

transmission of B. burgdorferi from feeding ticks (103). Specifically, migration to tick salivary

glands and spirochete multiplication in the feeding tick were disrupted due to passive

immunization with anti-OspA antibodies. These results suggest that a pre-established immune

response against OspA may prevent the transmission of B. burgdorferi from feeding ticks.

Further experiments also found that a high titer of passive OspA immunization was necessary to

expunge the spirochetes from a feeding tick, but a significantly lower titer was nevertheless

protective against infection in the mouse model (104). These studies eventually led to the

development of the first FDA-approved vaccine against B. burgdorferi, LYMErix, which used

recombinant, lipidated OspA as its immunogen (105, 106). To compensate for the noted

heterogeneity of the OspA protein, the most common North American OspA sequences were

used as the basis for the vaccine (107). Initally, the outlook for LYMErix was promising.

18

However, some reports of neurological and rheumatological adverse effects were reported by

users. Although it was determined that these adverse events of OspA vaccination were below

background based on a comparison with the clinical trials, the subsequent unpopularity of the

vaccine and the need for yearly booster vaccines led to its voluntary withdrawl in 2002 (108,

109). Investigation of the OspA epitope has revealed that the potential for cross-reactivity exists

between it and the immune cell surface molecule LFA-1, which could promote the development

of autoimmunity (110). The correlation of B. burgdorferi infection and autoimmune disorders

has been previously observed in individuals with certain HLA-DR alleles (e.g. HLA-DR4) (111).

These results suggest that certain genetic backgrounds are more susceptible to OspA-promoted

autoimmunity, and this hypothesis is being addressed in the future generation of OspA-based

vaccines through the engineering of OspA sequences to lack the molecular mimicry of the LFA-

1 epitope (112). In addition, investigations into chimeric OspA sequences are being conducted

so as to improve protection against multiple B. burgdorferi serotypes (113). Still other proteins

are currently being investigated as potential vaccinogens. OspC, a lipoprotein essential for the

transmission of B. burgdorferi to vertebrates, elicits an early, type-specific antibody response in

vivo, suggesting potential role as a human immunogen (114). In contrast to OspA, however, is

the diversity of OspC serotypes (115, 116). This remarkable diversity impedes development of

OspC-based vaccines by preventing broad coverage based on immunization with a single

serotype of the lipoprotein. To overcome this, chimeric OspC lipoproteins have been

constructed based on conserved regions between different serotypes and are undergoing

investigation for potential as vaccinogens (117, 118). These have the potential to convey broad

coverage against B. burgdorferi without the pitfalls of OspA vaccines, although further testing is

still needed to assess their efficacy in human subjects. Finally, other researchers are

19

investigating the novel avenues of tick salivary gland proteins or ΔospAB B. burgdorferi cell

preparations to induce protective immune responses (119, 120). In any case, it is undeniable that

an anti-Lyme vaccine is needed and that future research should focus on development of

prophylaxes against this disease.

20

Chapter 2: Lipoprotein Biosynthesis in B. burgdorferi

The understanding of lipoprotein biosynthesis in B. burgdorferi is primarily based on that

of E. coli. Like B. burgdorferi, E. coli is a diderm bacterium that localizes lipoproteins to both

its outer and inner membranes (25, 26, 121). Although E. coli does not express the multitude of

surface lipoproteins characteristic of B. burgdorferi, recent studies have shown that some

lipoproteins are indeed surface-exposed in this organism (122-126). The primary biochemical

research in which the function of various biosynthetic enzymes was also conducted, for the most

part, in E. coli. Therefore, the lipoprotein biosynthetic pathway will be described using E. coli as

a model and, where appropriate, clarifications will be made for differences observed in B.

burgdorferi. The lipoprotein biosynthesis genes in B. burgdorferi are given using the standard

nomenclature in parentheses following the accepted E. coli common name (31, 32, 127).

Lipoproteins are first translated in the cytoplasm without lipid modification. The

translated protein contains an N-terminal signal sequence, a characteristic region of amino acids

that directs the unfolded protein to the Sec translocase for export into the periplasm. In some

bacteria, certain lipoproteins are translocated in a folded conformation via the Tat (Twin arginine

translocation) machinery; however, this system appears to be absent from B. burgdorferi and will

not be discussed further (31, 32, 128). The signal sequence region typically contains three sub-

regions: an N-terminal region containing positively charged amino acids, a central hydrophobic

region with uncharged amino acids, and a C-terminal region containing a characteristic

consensus sequence termed the “lipobox” (25, 26). In E. coli, the lipobox consensus sequence is

typically Leu-[Ala/Ser]-[Gly/Ala]-Cys, with the final cysteine absolutely conserved in all

lipoproteins and acting as the site of lipid modification (25, 26). B. burgdorferi, however, has

been found to contain a much more degenerate lipobox (23). Based on comparisons of

21

experimentally verified lipoproteins, the B. burgdoreri lipobox likely includes one additional

amino acid at the N-terminal side and tolerates a larger variety of residues at each position with

the exception of the final cysteine residue, which is absolutely conserved in all lipoproteins.

This degeneracy of the B. burgdorferi lipobox has complicated in silico calculation of the

lipoprotein proteome (lipoproteome), and the identity of the B. burgdorferi has yet to be

conclusively established.

After translation, the unmodified lipoprotein peptide (“preprolipoprotein”) is then

directed to the Sec translocase for secretion to the periplasm (Figure 1) (25, 26, 121). Evidence

exists that, in E. coli, lipoproteins are exported by Sec in a co-translational manner mediated by

signal recognition particle (SRP; BB0694) and the inner membrane SRP receptor FtsY (BB0076)

(129). The Sec translocase is composed of a primary heterotrimer of the channel-forming,

integral inner membrane proteins SecY (BB0498), SecE (BB0395), and SecG (BB0054). The

ATPase SecA (BB0154) provides the driving force for protein translocation through ATP

hydrolysis. In addition, the integral membrane protein YidC (BB0442) assists with the lateral

transfer of the hydrophobic signal sequence region into the inner membrane, anchoring the

preprolipoprotein to the outer leaflet of the inner membrane (129). The accessory factors SecD

(BB0652), SecF (BB0653), and YajC (BB0651) form an inner membrane complex that serves to

assist protein translocation by coordingating YidC and SecYEG (130). These accessory proteins,

unlike the other members of the Sec translocase, are nonessential (121). In addition, certain

other bacteria (primarily the α/β/γ-proteobacteria) express the soluble cytosolic chaperone SecB

(no homolog in B. burgdorferi) that presumably serves to prevent premature folding in proteins

that are secreted post-translationally (131). Its absence from B. burgdorferi, and indeed many

other bacterial classes, suggests that other means of preventing premature folding of secreted

22

proteins have evolved. The necessity of these genes in B. burgdorferi is suggested indirectly

through transposon mutagenesis assays; no cells were recovered containing disruption of any of

the above listed components of the Sec translocase (37). This suggests that, in contrast to E. coli,

the SecDFYajC complex is essential in this organism.

After translocation, the preprolipoprotein is anchored at the outer leaflet of the inner

membrane through the hydrophobic region of its N-terminal signal sequence. The

preprolipoprotein is then modified through the addition of diacylglycerol to the thiol group of the

absolutely conserved cysteine by a thioether linkage. This reaction is catalyzed by the inner

membrane protein prolipoprotein diacylglyceryl transferase (Lgt; BB0362), with the

diacylglycerol group derived from phosphatidylglycerol, an abundant membrane phospholipid

(Figure 2) (132). The prolipoprotein is then liberated of its signal peptide through the action of

lipoprotein signal peptidase (Lsp; Signal Peptidase II; BB0469). Lsp cleaves the prolipoprotein

on the N-terminal side of the acylated cysteine, leaving the lipoprotein still embedded in the

membrane through its diacylglycerol modification. It has been shown that cleavage of the signal

sequence peptide is dependent on protein modification by Lgt, possibly due to the action of the

diacylglycerol group in coordinating the Lsp-prolipoprotein interaction (133). E. coli Lsp is

reversibly and noncompetitively inhibited by the cylic peptide antibiotic globomycin, preventing

signal peptide processing and subsequent export of outer membrane lipoproteins (134, 135).

Although data are limited, there is evidence that globomycin similarly inhibits Lsp homologs in

the genus Borrelia, lending support to the notion that lipoprotein biogenesis proteins are highly

conserved in diderm bacteria (136).

Cleaved signal peptides are then digested by an inner membrane protease, though the

identity of this protease is currently unclear. Initally, the atypical serine protease SppA (no

23

Figure 1. Sec-dependent translocation of lipoproteins. Secretion of lipoproteins occurs co-translationally through the binding of signal recognition particle (SRP) to the N-terminal hydrophobic signal peptide (121, 129, 130). Translocation occurs through the SecYEG channel and is driven by SecA-catalyzed ATP hydrolysis. Insertion of the lipoprotein’s signal peptide into the inner membrane is mediated by the integral membrane protein YidC, which associates with SecYEG through the SecDFYajC complex. IM = “inner membrane.” LP = “lipoprotein.” SP = “signal peptide.”

24

IM SecYEGSecDFYajC YidC

SecA

Ribosome

ATP

ADP

SRP

LP

SP

25

homolog in B. burgdorferi) was thought to be responsible for this digestion, as it was shown that

SppA, also called Protease IV, could digest signal peptides in detergent extracts (137). Later

studies instead pointed to RseP (BB0118), a member of the site-2 metalloprotease family I-CLiP,

as the protein responsible for signal peptide degradation (138). RseP also plays a role in the

regulation of the extracytoplasmic stress response through targeted cleavage of RseA (no

homolog in B. burgdorferi), the anti-sigma factor for E. coli σE (139). While sppA is not an

essential gene, rseP (yaeL/ecfE) is only dispensable when rseA is also deleted (140, 141). These

results suggest that the accumulation of cleaved signal peptides may be tied to the bacterial

extracytoplasmic stress response, and that multiple proteins may ultimately be responsible for

degradation of cleaved signal peptides. The rseP gene could be disrupted by transposon

mutagenesis in B. burgdorferi, suggesting that this gene is nonessential for in vitro growth (37).

This is not surprising, as the B. burgdorferi genome does not contain RseP’s cognate RseA nor

does it contain a σE homolog, with only three (rpoD (BB0712), rpoN (BB0450), rpoS (BB0771))

sigma factor genes present in the genome (31, 32, 142). The dispensability of rseP in B.

burgdorferi suggests that this organism possesses other, undiscovered means of degrading

cleaved signal peptides.

The final step of lipoprotein biosynthesis is acylation of the newly liberated amino group

on the N-terminal S-acylated cysteine through an amine bond (143). This is performed by the

inner membrane protein apolipoprotein N-acyltransferase (Lnt; BB0237) using

phosphatidylglycerol as the preferred acyl donor, although other phospholipids can be utilized

(144). This final acylation step is primarily only found in Gram-negative bacteria, although

exceptions do exist (notably, Mycobacterium) (145). Acylation by Lnt is a prerequisite for

release of lipoproteins from the inner membrane, as in depletion of the otherwise-essential Lnt

26

protein in E. coli results in lethal retention of Lpp (no homolog in B. burgdorferi) in the inner

membrane (146). Subsequent studies found that deletion of lnt could be tolerated by E. coli only

when both the lipoprotein localization complex LolCDE was overexpressed and either Lpp or

Lpp-peptidoglycan transpeptidases were absent (147). The other genes of lipoprotein

biosynthesis, lgt and lsp, have likewise been found to be essential in E. coli (140). Interestingly,

the gene for lnt is not essential in the Proteobacteria Francisella tularensis and Neisseria

gonorrhoeae (148). The deletion of lnt in these bacteria had no major effects on cell physiology,

suggesting that lipoprotein transport to the outer membrane was not dependent on N-acylation.

The authors of this study suggest that the dispensability of lnt in these bacteria is linked to

expression of a homodimeric, hybrid LolC/LolE protein (termed “LolF”, see next paragraph) and

speculate that bacteria expressing this hybrid protein may not require N-acylation of their

lipoproteins. Regulation of lipoprotein processing could also be a means by which pathogenic

bacteria regulate interactions with their host, a hypothesis which merits further inquiry in future

studies (149). B. burgdorferi contains two distinct genes for LolC and LolE; therefore, it is

believed that all lipoproteins are N-acylated and that this step is required for release of

lipoproteins to the outer membrane. Supporting this are transposon mutagenesis experiments

showing that no mutants could be recovered when lgt, lsp, or lnt were disrupted (37).

Lipoproteins are initially all situated at the outer leaflet of the inner membrane following

the final processing step by Lnt. In order for these proteins to perform their proper biological

function, they must then be sorted to an appropriate destination. In E. coli, most lipoproteins are

either sorted to either the inner leaflet of the outer membrane or retained in the outer leaflet of

the inner membrane, although some exceptions have recently been found (126). Proper sorting

of lipoproteins is key to their biological role in the cell and, indeed, lipoprotein mislocalization

27

Figure 2. Post-translational modification of lipoproteins. After Sec-mediated secretion, lipoproteins undergo a sequential series of modifications in which the absolutely conserved N-terminal cysteine residue is lipidated and the N-terminal signal peptide is removed (25, 26, 143, 147). OM = “outer membrane.” IM = “inner membrane.” LP = “lipoprotein.” SP = “signal peptide.”

28

IM

OM

LP

SP

Lgt Lsp

LP

SP

LP

Lnt

LP

29

can often prove detrimental to bacterial viability. In E. coli, for example, mislocalization of the

major outer membrane lipoprotein Lpp to the inner membrane as a result of lnt deletion is toxic

to the cell due to aberrant peptidoglycan crosslinking (147). Sorting of lipoproteins to the outer

membrane is accomplished by the Lol machinery (25, 26, 121). The Lol sorting machinery is

composed of an inner membrane protein complex of LolCDE, the periplasmic lipoprotein

chaperone LolA, and the outer membrane lipoprotein acceptor LolB. Initially, lipoproteins either

associate with the inner membrane LolCDE complex or “avoid” association with it based on

whether the lipoprotein is destined to sort to the outer membrane or remain in the inner

membrane. In E. coli, discrimination between inner and outer membrane lipoproteins is

achieved through recognition of amino acid residues immediately following the acylated

cysteine. Initially, it was found that an aspartic acid at the “+2” position, where the acylated

cysteine represents “+1”, causes lipoprotein retention in the inner membrane (150). It was later

discovered that the amino acid identity at +3 can also affect inner membrane retention of

lipoproteins in E. coli (151, 152). The mechanism behind this retention has been shown to be

possibly the result of steric and electrostatic interactions of the +2 and +3 amino acids with the

abundant membrane phospholipid phosphatidylethanolamine (153). Similar studies in the

related bacterium Pseudomonas aeruginosa showed that the amino acids at +2/+3/+4 could all

influence lipoprotein sorting in this organism (154, 155). However, in B. burgdorferi results

have shown that retention of inner membrane lipoproteins is not strictly dependent on the amino

acid identities at +2/+3/+4, and the exact logic by which B. burgdorferi retains lipoproteins in the

inner membrane remains unknown (27-30, 156). In E. coli, the complex LolCDE is composed of

the integral inner membrane proteins LolC (BB0078/0079, region originally mis-sequenced in

B31) and LolE (BB0081), as well as a homodimer of the ATPase LolD (BB0080). The

30

homodimer of LolD binds two ATP molecules on the cytoplasmic face of the inner membrane,

utilizing ATP hydrolysis to drive the transfer of lipoproteins from the inner membrane to the

periplasmic chaperone LolA through the actions of LolC and LolE (Figure 3) (157). The

proteins LolC and LolE show remarkable similarity to each other; however, despite their

similarity they seem to play distinct roles in the cell (158). Site-directed crosslinking studies

have shown that LolE is responsible for binding lipoproteins at the inner membrane whereas

LolC binds LolA in the periplasm, with ATP hydrolysis driving a conformational change in the

proteins that results in “mouth-to-mouth” transfer of lipoproteins from LolE to LolC and, finally,

to LolA in the periplasm (159, 160). Studies in E. coli have shown that expression of the

LolCDE complex is essential for cell viability, and that disruption of the complex results in the

lethal accumulation of lipoproteins at the inner membrane (161). Likewise, transposon

mutagenesis studies in B. burgdorferi have supported the hypothesis that expression of LolCDE

is necessary for cell survival (37).

The periplasmic chaperone LolA (BB0346) is responsible for transport of lipoproteins

between the inner and outer membranes. Originally termed “p20”, it was first discovered in E.

coli as the factor responsible for the release of soluble Lpp from spheroplasts (162). The solved

crystal structure of LolA revealed that the protein contains a distinct hydrophobic cavity formed

by an 11-stranded, antiparallel β-sheet in an unclosed β-barrel configuration with an α-helical

“lid” (163). This hydrophobic cavity was proposed to contain the insoluble lipid moiety of the

cargo lipoproteins, although to this date to LolA-lipoprotein co-crystals have been achieved. A

subsequent crystallization of the P. aeruginosa revealed that it contains additional hydrophobic

surface patches that, when mutated, reduce or eliminate lipoprotein transport function of LolA

(164). The authors speculate that the surface hydrophobic patches of P. aeruginosa may

31

Figure 3. The sorting of lipoproteins via Lol machinery. Lipoproteins, after lipidation of the conserved cysteine residue and cleavage of the signal peptide, are sorted through the inner membrane LolCDE complex, the periplasmic chaperone LolA, and the outer membrane receptor LolB (25, 26, 157, 162, 165). Lipoproteins either interact with the LolCDE complex or avoid the complex based on bacterial species-specific sorting signals as described in the text.

32

IM

OM

LP

LolC LolE

LolD LolD

LolA LP

LolB

LP

ATP ADP

33

accommodate lipoprotein acyl chains, and that the entire lipid moiety may not be contained

within the central hydrophobic cavity. In E. coli, LolA is essential for the survival of the

bacterium and its depletion results in the toxic buildup of lipoproteins at the inner membrane

(166). In B. burgdorferi, the homolog of LolA appears to be likewise essential for cell viability

(37).

Integration of lipoproteins into the outer membrane is achieved by transfer from LolA to

the outer membrane lipoprotein acceptor LolB (no homolog in B. burgdorferi), which is itself a

lipoprotein (165). Depletion of LolB was lethal for cells and prevented the incorporation of

outer membrane lipoproteins into E. coli total membrane fractions. Further, a soluble form of

LolB was found to be able to accept lipoproteins from LolA in vitro. These results also show

that lipidation of LolB is disensable for transfer of lipoprotein from LolA in vitro. Further

experimentation revealed that soluble periplasmic LolB could compensate for loss of lipidated

LolB in vivo, with the corresponding localization of lipoproteins to the outer membrane (167).

Transient mislocalization of outer membrane lipoproteins to the inner membrane was noted,

though, and showed that soluble LolB incorporated LolA-delivered lipoproteins into cell

membranes indiscriminately. These results show that lipidation of LolB is dispensable for its

function and primarily serves to assist LolB in properly integrating lipoproteins into the outer

membrane. Site-directed crosslinking studies have shown that LolA transfers lipoproteins to

LolB in a “mouth-to-mouth” fashion, wherein the lipid moiety moves from one hydrophobic

cavity to the other while being constantly shielded from the hydrophilic periplasm (160). LolB

has been crystallized in a soluble form and shows distinct structural similarity to LolA, despite a

notable lack of primary sequence homology (163). It too has an unclosed β-barrel structure

composed of 11 antiparallel β-strands and an α-helical lid, with a distinct hydrophobic cavity that

34

accepts the lipid group of transported lipoproteins from LolA. However, structural comparison

of crystallized LolA and (soluble) LolB proteins shows that the central cavity of LolB is more

hydrophobic than that of LolA (163). This difference in hydrophobicity results in an irreversible

transfer of lipoproteins from LolA to LolB due to the difference in affinity of the lipid moiety for

the two hydrophobic cavities (168). This allows for an energy-independent transfer of the

lipoprotein cargo, a necessity in the ATP-devoid periplasm. Other structural differences between

LolA and LolB also contribute to their different functions (169). The mechanism by which LolB

releases bound lipoproteins and integrates their lipid moiety into the outer membrane, however,

is currently unknown.

Unlike the other members of the Lol machinery, which are widely conserved in Gram-

negative bacteria, LolB seems to be found only in the β- and γ-proteobacteria (170). This raises

the interesting question of how other bacteria integrate lipoproteins into their outer membrane, as

the insolubility of the lipid modification of lipoproteins implies that some outer membrane

receptor must accept the lipid moiety from LolA. In B. burgdorferi, the abundance of outer

membrane lipoproteins suggests that a functional homolog of LolB exists (27). The identity of

this outer membrane lipoprotein receptor in B. burgdorferi, and whether it can be found in other

bacteria that lack an identifiable lolB gene, is currently under investigation. Studies of soluble

periplasmic LolB in E. coli have shown that it could at least somewhat compensate for the

absence of lipidated, outer membrane LolB in vivo (167). These results suggest that soluble

periplasmic factors can receive lipoproteins from LolA and that future searches for the receptor

of LolA-lipoprotein complexes should not be limited solely to outer membrane proteins.

The majority of lipoproteins in E. coli are localized to the periplasmic face of the outer

membrane or retained at the inner membrane. However, recent evidence suggests that surface

35

Figure 4. Alignment of the B. burgdorferi OppA homolog tether sequences. The tether sequences of the five OppA homologs were aligned using the Clustal Omega program using the default settings (173). The OppAIV (BB_B16) tether was defined based on its crystal structure, PDB# 4GL8. Other OppA tethers were extrapolated based on the OppAIV tethers. OppA homologs are annotated using the standard B. burgdorferi genetic locus notation (31, 32).

36

37

exposure of lipoproteins is a more common phenomenon than once thought. The major outer

membrane lipoprotein Lpp has been known for some time to exist in both a peptidoglycan-bound

form and an unbound form, and it was recently shown that the unbound form spans the periplasm

and is exposed on the bacterial surface (122). This was the first recorded example of a

membrane lipoprotein possessing two distinct cellular localizations. Subsequent studies also

found that the membrane lipoprotein RcsF (no homolog in B. burgdorferi), the sensor protein of

the Rcs envelope stress response system, was also found to adopt a unique bi-localization

phenotype (123, 171). RcsF, after localization to the inner leaflet of the outer membrane, is

“threaded” through the lumen of several β-barrel outer membrane proteins during assembly by

the Bam Complex. This results in RcsF becoming localized on the bacterial surface at its N

terminus, while the C-terminal signaling domain remains in the periplasm. RcsF seems to be able

to utilize different outer membrane proteins, with interactions detected between RcsF and

OmpA, OmpC, and OmpF. Finally, the pathogen Neisseria meningitidis expresses two

noteworthy outer membrane proteins termed surface lipoprotein assembly modulators (Slam; no

homolog in B. burgdorferi) (172). Slam proteins were shown to be responsible for export of the

outer membrane lipoprotein TbpB to the bacterial surface and, when expressed in E. coli, Slam

proteins were sufficient to reconstitute surface lipoprotein secretion. Conservation of Slam

proteins in Proteobacteria genomes was also reported by the authors. Preliminary analysis of the

B. burgdorferi genome shows that it does not contain an identifiable homolog to Slam. Because

of the abundance of surface lipoproteins in B. burgdorferi, it would not be surprising for another

membrane protein to be responsible for export of lipoproteins to the bacterial surface.

38

Chapter 3: Previous Research of B. burgdorferi Lipoproteins

Although the E. coli proteins involved in the biosynthesis have identifiable homologs in

B. burgdorferi (with the notable exception of LolB), lipoprotein sorting appears to obey a

completely different set of rules in this spirochete than in Proteobacteria (27-30, 156). Notably,

B. burgdorferi does not follow the above described +2/+3/+4 rule that is present in other

bacteria. A review of previously localized lipoproteins shows that residues in these positions are

highly variable and residues seem to be common between inner membrane, outer membrane sub-

surface, and surface lipoproteins (27-30). It has also been shown that, in contrast to the

functional regions of the protein, the N-terminal region can be highly variable even in

homologous lipoproteins. For example, an alignment of the five OppA homologs in B.

burgdorferi shows significant variability in the +2 through +9 positions, in contrast to the

conservation seen in the C-terminal regions of the proteins (Figure 4). As this N-terminal region

was previously thought to contain the information necessary for lipoprotein sorting, this

variability suggests that other, unknown factors may dictate sorting. Finally, mutagenesis

experiments of the prototypical surface lipoproteins OspA and OspC have shown that, although

deletion or mutagenesis of tether residues C-terminal from the +2/+3/+4 positions can modulate

surface localization of these lipoproteins, their export to the outer membrane remains largely

unaffected through tether mutations (28-30). Currently, it is not well understood why inner

membrane lipoproteins are retained and not exported to the outer membrane, although

interesting, testable hypotheses can be formulated based on existing data.

In contrast to sorting at the inner membrane, surface exposure of outer membrane

lipoproteins has been characterized to a fuller extent. Mutagenesis assays have shown that

alteration of key residues in OspA and OspC, residues in so-called “essential tether” motifs, can

39

cause sub-surface retention of an otherwise wild-type protein. In addition, deletion of OspC

tether regions C-terminal of the essential tether can likewise cause subsurface retention of the

lipoprotein. These results suggest that the N terminus of a mature lipoprotein plays a role in

surface localization and that alteration of this sequence can modulate a lipoprotein’s surface

exposure (28-30). In addition, experiments involving fusion of OspA to calmodulin, a protein

whose folding is dependent on the binding of Ca2+, revealed that OspA containing “sub-surface”

tether mutations could be exported to the surface in the context of an unstructured C terminus

(174). In addition, a separate study found that a sub-surface mutant of OspA could be restored to

the bacterial surface through fold-destabilizing mutations at the C terminus, indicating that

lipoprotein secretion through the outer membrane may initiate at the C terminus (29). These

results, taken together, suggest that lipoprotein surface exposure is an interplay between

recognition of an N-terminal export signal and sufficient disorder at the C terminus. It has also

been proposed that the driving force for export of lipoproteins to the surface is the potential

energy gradient of protein folding outside of the cell, and that the N-terminal region is crucial in

binding holding chaperones that prevent premature folding in the periplasm (25). This model

would explain how lipoproteins are exported out of the periplasm in the absence of molecular

energy sources such as ATP, as well as accounting for the retention of surface lipoprotein tether

mutant that show sub-surface, premature folding (29, 174). However, the identity of the putative

holding chaperone, the precise structure-function of the B. burgdorferi Lol homologs, the

identity of a potential outer membrane LolA-lipoprotein complex receptor, and the outer

membrane factors responsible for lipoprotein surface exposure all remain a mystery. Although

evidence exists in E. coli that the outer membrane β-barrel assembly complex Bam can cause

surface localization of lipoproteins by folding outer membrane proteins around them (see RcsF

40

in 1.3 The Fundamental Role of B. burgdorferi Lipoproteins, above), there is currently no

evidence to support this function of the Bam complex in B. burgdorferi.

The development of a powerful “toolbox” of genomic and proteomic techniques,

however, has put the field of B. burgdorferi research in a place to finally answer these questions.

Although B. burgdorferi is not nearly as experimentally tractable as the model organism E. coli,

endeavoring investigators have discovered ways to manipulate the bacterium in order to

overcome many of the limitations inherent to the spirochete. B. burgdorferi can be cultured in

vitro to high densities using artificial culture medium, in contrast to certain other spirochetes

such as Treponema pallidum (175-177). B. burgdorferi can also be transformed with foreign

DNA through electroporation, and the development of E. coli-B. burgdorferi shuttle vectors has

facilitated cloning of spirochetal genes (178, 179). Techniques to localize proteins in B.

burgdorferi are well-established and include surface “shaving” using a membrane-impermeable

protease and fractionation of the outer membrane of the cell from the protoplasmic cylinder (180,

181). Analysis of the products of these assays is accomplished by Western Blot using antisera

specific to characteristic proteins (27-30, 156). Finally, it was recently shown that B. burgdorferi

detergent extracts of surface shaving assays are amenable to proteomics analysis by

Multidimensional Protein Identification Technology (MudPIT) (27). This demonstrates that

existing protocols can be applied to emerging, high-throughput technologies to expedite

characterization of the B. burgdorferi proteome.

41

Chapter 4: Localization of B. burgdorferi Lipoproteome

(Note: An abridged version of this chapter was published as: Dowdell AS, Murphy MD, Azodi

C, Swanson SK, Florens L, Chen S, Zückert WR. Comprehensive Spatial Analysis of the

Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial

Surface. J Bacteriol. 2017 Feb 28;199(6). pii: e00658-16. doi: 10.1128/JB.00658-16. PubMed

PMID: 28069820)

4.1 Rationale for Study

It has been known for some time that certain proteins are indispensable for infection of

vertebrates by B. burgdorferi (182). For example, the protein OspC is required to establish an

infection in the mouse model of Lyme borreliosis, and the genetic locus containing the antigenic

variation protein VlsE is necessary for persistence in immunocompetent mice (71, 82). It was

also discovered early on that many of these critical proteins are lipoproteins and are

immunogenic in humans (46, 99). Therefore, a significant portion of the past few decades of

research has been focused on the clinical importance of lipoproteins in the prevention and

treatment of Lyme borreliosis (112-114, 117, 118). These efforts yielded the first FDA-

approved, anti-Lyme vaccine (LYMErix) at the end of 1998 (101, 105, 106). This vaccine was

based on immunization against the B. burgdorferi lipoprotein OspA, which is expressed in the

unfed tick prior to influx of the blood meal. However, the vaccine’s perception by the public

was tainted by reports of vaccine-related arthritis and other complications (108, 109). While the

CDC and FDA evaluated all available data and concluded that no correlation exists between the

vaccine and the reported adverse events, the vaccine was nevertheless perceived negatively by

the public and sales declined. In addition, it was found that high anti-OspA titers are required in

42

order to prevent B. burgdorferi transmission, further complicating adoption of LYMErix. For

these reasons, the vaccine was voluntarily removed from the market in 2002, just a few years

after its introduction.

Previous research has focused on just a limited subset of lipoproteins, with the focus on

those that have a clear role in pathogenesis or application as a possible vaccinogen. This has left

many lipoproteins under-studied, aside from cursory in silico homology analysis. One

complication to the further study of B. burgdorferi lipoproteins is that, due to their comparatively

degenerate lipobox region, it is difficult to predict lipoproteins based from primary sequence

alone. The “gold standard” for identifying protein lipid modification, the detection of

incorporated [3H]-palmitate into an immunoprecipitated protein, is low-throughput and ideally

requires antisera to the protein of interest (46). As such, study of B. burgdorferi lipoproteins

focused on a handful of “usual suspects” for some time and neglected a potentially large fraction

of the remaining B. burgdorferi proteome.

In 2006, an algorithm was developed specifically for prediction of lipoproteins in

spirochetal genomes. Utilizing experimentally verified lipoproteins as a training set, the SpLip

algorithm identified the putative “lipoproteome” of the B. burgdorferi type strain B31 (23).

Many of the proteins identified as lipoproteins in this study were “under-studied” and lacked the

in-depth characterization that had been performed for other lipoproteins such as OspA. Using

this in silico prediction, our group hypothesized that we could perform an initial characterization

of this putative lipoproteome through localization of each protein to a specific cellular location.

Based on our previous observation that lipoproteins seem to be secreted to the surface by default,

we hypothesized that the majority of lipoproteins would be surface-localized (30). We also

hypothesized that homologous lipoproteins would localize similarly, such as those encoded by

43

Figure 5. Plasmid map of the B. burgdorferi lipoprotein expression library vector backbone pSC-LP. pSC-LP is a derivative of the B. burgdorferi-E. coli shuttle vector pBSV2 (29, 179) that drives expression of cloned genes using the B. burgdorferi flaB promoter (PflaB) and provides a C-terminal hexa-histidine tag preceded by a flexible 6-amino-acid linker peptide. Restriction enzyme sites and primer sequences used for amplification and cloning of lipoprotein genes are indicated (see also Table 1 and text).

44

45

the evolutionarily related cp32 family of plasmids (100, 183). Finally, we hypothesized that,

after localizing the entire lipoproteome, sorting rules for B. burgdorferi could be established

similar to those in Proteobacteria (150, 154).

4.2 Experimental Design

(Note: An unabridged version of experimental protocols can be found in Chapter 5)

4.2.1 Bacterial Strains and Culture Conditions

E. coli strains TOP10 and DH5α (Invitrogen) were used for plasmid cloning and

propagation. E. coli was grown in LB-Miller broth or on LB-Miller agar plates (both from BD

Difco) at 37°C, supplemented with 50 µg/mL kanamycin (Sigma-Aldrich) as necessary. B.

burgdorferi strain B31-e2, a high-passage noninfectious clone of the type strain B31 (95)

(provided by Dr. Brian Stevenson, Univ. of Kentucky, Lexington, KY), was chosen due to its

amenability to transformation and established use in lipoprotein localization assays (28-30).

Additionally, low-passage infectious B. burgdorferi B31-A3 (184) (provided by Dr. Patti Rosa,

NIH/NIAID Rocky Mountain Laboratories, Hamilton, MT) was used for proteomic analysis of

cellular protein fractions by Multidimensional Protein Identification Technology (MudPIT) mass

spectrometry. B31-A3 was confirmed to contain all linear and circular plasmids characteristic of

strain B31 with the exception of lp5, using a set of multiplex-PCR-compatible oligonucleotide

primers (185) (data not shown). B. burgdorferi B31-e2 and B31-A3 were maintained in BSK-II

complete medium at 34°C, containing 300 µg/mL kanamycin as necessary (175, 177). Recovery

of B. burgdorferi transformants was performed using semi-solid BSK-II as described, with plates

incubated at 34°C in a humidified 5% CO2 atmosphere until the development of colonies (175,

177).

46

4.2.2 Construction of an epitope-tagged B. burgdorferi lipoprotein expression library

A total of 127 B. burgdorferi type strain B31 lipoproteins were selected for the study

based on the cumulative list of “probable”, “possible” and “false negative” lipoprotein genes

identified by the SpLip algorithm (23) (Table 2); of note, this algorithm-based list omits some

lipoproteins such the variable surface lipoprotein VlsE (81). Genomic DNA from cultured low-

passage infectious B. burgdorferi B31-A3 was isolated (Promega Wizard gDNA Kit), and

lipoprotein genes were amplified by PCR using Phusion HF enzyme (New England Biolabs)

with gene-specific oligonucleotide primers (Integrated DNA Technologies) containing 5’ NdeI

and XmaI restriction site extensions, respectively (Table 1). The resulting PCR amplicons were

digested with NdeI and XmaI and ligated into pSC:LP (Figure 5), a vector backbone derived

from recombinant plasmid pSC1000 (29) by digestion with NdeI and XmaI. pSC1000, a

derivative of the B. burgdorferi-E. coli shuttle vector pBSV2 (179), expresses the lipoprotein

OspA (BBA15) under the constitutive flagellin promoter (PflaB) with a C-terminal his-tag; the

NdeI (CA’TATG) and XmaI (C’CCGGG) sites are within the start codon and the his-tag linker,

respectively. Due to Borrelia DNA being about 70% AT, this approach allowed for the direct

amplification by PCR and in-frame cloning of 115 Borrelia lipoprotein genes. 10 lipoprotein

genes containing internal NdeI sequences were amplified and cloned using a modified forward

PCR primer that produced an amplicon with a 5’ blunt-end compatible with the pSC:LP NdeI

site filled in using Klenow (Figure 5). BB0352 and BBB16 (oppA4) were cloned under their

native promoters, taken to be within about 150 bp upstream of their respective start codons.

Recombinant plasmids were recovered from cultured E. coli transformants using the QIAprep

Spin Kit according to the manufacturer’s instructions (QIAGEN) and screened for the expected

47

Table 1. List of oligonucleotide primers used for B. burgdorferi lipoprotein amplification. Oligonucleotide sequences used to amplify and clone lipoprotein genes as described in the text.

48

ORF Cloning Primer Fwd Cloning Primer Rev BB_A15 N/A - this is pSC1000 N/A - this is pSC1000 BB_0365 ACCTGCATATGTATAAAAATGGTTTTTTTAA

AAAC ATCAGCCCGGGATTCGTTAACATAGG

TGAAATTTT BB_B19 ACCTGCATATGAAAAAGAATACATTAAGTG

C ATCAGCCCGGGAGGTTTTTTTGGACTT

TCTGC BB_B16 CATGGAGGAATGACATATGAAAATATTGAT

AAAAAAGTTAAAAG ATCAGCCCGGGTTTAATTGGTTTTATT

TCAGATAAATT BB_0328 ACCTGCATATGAAAAAGGAAAATCCCATGA

AATAT ATCAGCCCGGGTTTTTTAGTTTTAATA

TCTTCATATAAAT BB_0329 ACCTGCATATGAAATTACAAAGGTCATTATT

TTTA ATCAGCCCGGGTTTATTTTTTAATTTT

AGCTGAGATA BB_0330 ACCTGCATATGAGCTTTAATAAAACTAAAA

AAATC ATCAGCCCGGGATTATGTTTTGCATTT

TTAATTGG BB_A24 ACCTGCATATGATTAAATGTAATAATAAAA

CTTTT ATCAGCCCGGGGTTATTTTTGCATTTT

TCATCAG BB_A16 GGAGGAATGACATATGAGATTATT CCCCGGGTTTTAAAGCGTT BB_P38 TGAATAAGAAAATGAAAATGTTTAT ATCAGCCCGGGTTTTAAATTTCTTTTA

AGCTCTTC BB_J09 ACCTGCATATGAAAAAATTAATAAAAATAC

TACTG ATCAGCCCGGGAGTATTTAACAAGGC

CACAAC BB_R42 ACCTGCATATGAATAAAAAAATAAAAATGT

TTATT ATCAGCCCGGGTTCTTTTTTACCTTCT

ACAGTTTC BB_S41 ACCTGCATATGAATAAGAAAATGAAAAATT

TAAT ATCAGCCCGGGTTTTTTATCTTCTATA

TTTTGAGG BB_B08 ACCTGCATATGAAAAAAAAGTTTAATTTTAT

TTTTC ATCAGCCCGGGTTGTAAAAATTTTTCA

ATTGC BB_0028 ACCTGCATATGAAACAAAAATACGAAAAC ATCAGCCCGGGTTCTTTAGTTAATTTT

CTGTTTTC BB_0689 ACCTGCATATGAAAAAATTGATTATAATTTT

TAC ATCAGCCCGGGATTCTTATATTTTCTT

TTTCCAA BB_0840 ACCTGCATATGAAGAATATTAATAGATTAA

TATTATT ATCAGCCCGGGACTTCCTGAGCACCA

G BB_B27 ACCTGCATATGAAGAAGTTTTTAATATCCG ATCAGCCCGGGAGTTAAAAACTTTTT

GAGTATATAT BB_0324 ACCTGCATATGAAAAAGCTAATATTACTAA

AC ATCAGCCCGGGTTTTTTATTATTTTCT

ATTTTATTTAAT BB_0832 ACCTGCATATGTTAAAAACATTAACAAAAA

TA ATCAGCCCGGGGGTTTGAATATTTGA

AGCTAG BB_0144 ACCTGCATATGTATAAATTATTTTTATTTTTT

ATTAT ATCAGCCCGGGATCAAATAAGGTCTT

ATATTTTTC BB_0382 ACCTGCATATGAGAATTGTAATTTTTATATT

CG ATCAGCCCGGGTAACTTTAATATTTGT

TTTATAAAAAT BB_0628 ACCTGCATATGAGAAAGTGTTTTGTTAG ATCAGCCCGGGATAAACTAATCTAAG

TTTTATTGG BB_0141 ACCTGCATATGAATTTGATTTTTAATATTAA

TTTAT ATCAGCCCGGGAATATTGCTTTCGGCT

G BB_B25 ACCTGCATATGAAATACTGTTTTTCTTTG ATCAGCCCGGGTAAAATTTTGTTTTTA

AATGCAT BB_0542 ACCTGCATATGGATAATTTAAATAGTATAA

ATAGT ATCAGCCCGGGACTCTTAAGAATAGA

AGAAAC BB_0227 ACCTGCATATGCTAAATCCAAGAACAA ATCAGCCCGGGTTTTACAAATTGTGCT

ATTTC

49

BB_0460 ACCTGCATATGAAAACTAGAATAATCATTTTTC

ATCAGCCCGGGAGACTGAGAGTGAAGG

BB_0298 ACCTGCATATGAAAATTTTGTGGTTAATAA ATCAGCCCGGGCTTCAAATTACTCATGATCC

BB_0215 ACCTGCATATGAAAAAAGTTATTATCTTAATTTT

ATCAGCCCGGGTGTTTTTATCCCTAAAAAGC

BB_0385 ACCTGCATATGTATTATGAGATCTATTTTTTG

ATCAGCCCGGGATTTTCCATTTGCAAAACA

BB_0323 TGAATATAAAGAATAAATTAATATCGC ATCAGCCCGGGTTTGGCAGGAATTATTATCTTC

BB_0664 ATAATCATATGTTACATAATACAATGC ATCAGCCCGGGTTGGGCTTTGATTTCTTG

BB_0398 ACCTGCATATGAATCTATTGGTCAAAATTG ATCAGCCCGGGTTTTTTACCTAAATACACGC

BB_0155 ACCTGCATATGAAAAAACACTATAAAGCTC ATCAGCCCGGGTAATTTTAATAATGCATTTAAATTCC

BB_0384 ACCTGCATATGTTTAAAAGATTTATTTTTATTAC

ATCAGCCCGGGTAACTTTGATTTAAATAAATCAAATG

BB_0158 ACCTGCATATGGTATTTAGAACATATAAAC ATCAGCCCGGGTATATCTTCAAATAAATCTCCAG

BB_0806 TGAAATTTGTTTTGAATAATTTATTTAAAG ATCAGCCCGGGTAACTGTTTTTTAATATTTTCATC

BB_0758 ACCTGCATATGCTGCAAAAAAGTATGG ATCAGCCCGGGATTGATAACTATTTTTTGAGG

BB_0844 ACCTGCATATGAAAAAAAAAAATTTATCAATTTAC

ATCAGCCCGGGTTTACTCGTCTCTAAAAAATC

BB_0193 ACCTGCATATGTCTGGCCCTAAAAAAC ATCAGCCCGGGTTGTTTATAAATGTTAAATTCATATTG

BB_0823 ACCTGCATATGAATACAAAAACATTATATTTAATA

ATCAGCCCGGGTTTAGAATGATCAATTATTAAATTG

BB_0652 ACCTGCATATGAAAAAAGGATCTAAGC ATCAGCCCGGGATTACTCTTTGCATATTTTGAAC

BB_0536 GGAGGAATGACATATGAATTATCAAAG TGAGCCCCCGGGTTCAGGTATTAG BB_0224 ACCTGCATATGCCTGGAAGAAAGG ATCAGCCCGGGATTGTATTGGTAATTA

AAGCA BB_0475 ACCTGCATATGATGAAAGCTCAAAAATTTA ATCAGCCCGGGTTGTTTTTCAAAAATG

TATAATG BB_0213 ACCTGCATATGCAGAGCGGATTAAA ATCAGCCCGGGGCCAACTTTAAAAAA

AAGATT BB_0171 ACCTGCATATGGGACGAAACTTTTTAG ATCAGCCCGGGCCAATCTTCTTTTGGA

ACAG BB_0456 ACCTGCATATGAAGCAAAAATTAAGTTG ATCAGCCCGGGAATTCCAGGCGATAA

AAC BB_0352 ACCTGCATATGAATAATTTTATGAGAATAA

AAAAT ATCAGCCCGGGATTTTTATTTTTTTTT

AGATTATTAGC BB_B09 ACCTGCATATGAAATACCTTAAAAACATTTC ATCAGCCCGGGAAATTTATGCCTACTT

GATTG BB_A62 ACCTGCATATGACAAAATTAATGTACGC ATCAGCCCGGGCTTTTTCATTGACTTT

GTC BB_A57 ACCTGCATATGAACGGCAAGCTTAG ATCAGCCCGGGTTGATAATTTTTTTCT

ACCAATAA BB_A33 ACCTGCATATGAAGAGATATATTTATGTATA

TATC ATCAGCCCGGGTTTTTGAATTAGTGCT

AAAGC BB_A34 ACCTGCATATGATAATAAAAAAAAGAGGAC ATCAGCCCGGGTTCTTCTATAGGTTTT

ATTTCTG

50

BB_A05 ACCTGCATATGAATAAAATAGGAATTGCAT ATCAGCCCGGGACGCCTGCTAATCTCTTTTA

BB_A59 ACCTGCATATGGTTAAAAAAATAATATTTATTTC

ATCAGCCCGGGATTGCTCTGCTCACTCTC

BB_A69 ACCTGCATATGAAAAAAGCCAAACTAAATAT

ATCAGCCCGGGATAAAAGGCAGATTGTAAAG

BB_A04 ACCTGCATATGAAAAGAGTCATTGTATCC ATCAGCCCGGGGTTAATTAACGAATTAAATGC

BB_A64 ACCTGCATATGAAGGATAACATTTTGAAA ATCAGCCCGGGCTGAATTGGAGCAAGAATA

BB_A07 ACCTGCATATGTGTGGGAGACGTATG ATCAGCCCGGGAAACGAAGCAGATGCATC

BB_A68 ACCTGCATATGAAAAAAGCCAAACTAAATAT

ATCAGCCCGGGGTAAAAGGCAGGTTTTAAAG

BB_A36 ACCTGCATATGATGCAAAGGATAAGTATT ATCAGCCCGGGAACATTTCCATAATTTTTCAAA

BB_A65 ACCTGCATATGAATAAAATAAAATTATCAATATTAAT

ATCAGCCCGGGATTATTATTAAATTTAAATAATGTGTC

BB_A32 ACCTGCATATGAGAATTACCGGTCTTC ATCAGCCCGGGAAGCTTTCTGTTTCTACG

BB_A03 ACCTGCATATGAAAAAAACGATTATTGTATT

ATCAGCCCGGGTATAGTGTCTTTAAGTTTATTAAGT

BB_A72 ACCTGCATATGCATAAGGAGAGTGTT ATCAGCCCGGGTTCATTTTTCTGATCTTTTAAC

BB_A14 ACCTGCATATGCAAATTAAAAATTTCCCC ATCAGCCCGGGAGGTATATTTTTTGAGTATTTTTTG

BB_P28 ACCTGCATATGAAAATCATCAACATATTATT ATCAGCCCGGGGGACCCATTGCCGCAG

BB_P39 ACCTGCATATGAATAAAAAAACAATTATTATTTG

ATCAGCCCGGGATCTTCTTCATCATAATTATCC

BB_P27 TGAGAAATAAAAACATATTTAAATT ATCAGCCCGGGATTAGTGCCCTCTTCGAG

BB_S30 ACCTGCATATGAAAATCATCAACATATTATTT

ATCAGCCCGGGGCCACCATTATTGCAGTT

BB_R40 ACCTGCATATGAATAAGAAAATGAAAAATTTA

ATCAGCCCGGGGCCAATACTTTGCTCATC

BB_R28 ACCTGCATATGAAAATTATCAACATATTATTTTG

ATCAGCCCGGGTGAATTTTTGCACGTACTAC

BB_M38 ACCTGCATATGGAGCAACTTATGAATAAG ATCAGCCCGGGTTCTTTTTTATTAGAATCTTTAGAT

BB_M28 ACCTGCATATGAAAATCATCAACATATTATT ATCAGCCCGGGGGAACCACCATTGTTGC

BB_M27 TGAGAAATAAAAACATATTTAAATT ATCAGCCCGGGATTAGTGCCCTCTTCGAG

BB_O39 ACCTGCATATGAATAAGAAAATGAAAATGTT

ATCAGCCCGGGTTCTTTTTTATCTTCTTCTATTC

BB_O40 ACCTGCATATGAATAAAAAAATATTGATTATTTTTG

ATCAGCCCGGGATATGAATTACTATCCTCAATG

BB_O28 ACCTGCATATGAAAATTATCAACATATTATTTTG

ATCAGCCCGGGATTGCAGGTAGCAGTTGC

BB_L39 TGGAGAAATTTATGAATAAGAA ATCAGCCCGGGTTTTAAATTTCTTTTAAGCTCTTC

BB_L40 ACCTGCATATGAATAAAAAAACAATTATTATTTG

ATCAGCCCGGGATCTTCTTCATCATAATTATCC

51

BB_L28 ACCTGCATATGAAAATCATCAACATATTATT ATCAGCCCGGGGGAACCGTTGCATGTAG

BB_N38 TGAATAAGAAAATGAAAATGTT ATCAGCCCGGGTTTTAAATTTTTTTTAAGCACTTC

BB_N39 ACCTGCATATGAATAAAAAAACATTGATTATT

ATCAGCCCGGGCTGACTGTCACTGATGTATC

BB_N28 ACCTGCATATGAAAATTATCAACATATTATTTTG

ATCAGCCCGGGTTGCTGAGCTTGGCAG

BB_C10 ACCTGCATATGCAAAAAATAAACATAGC ATCAGCCCGGGATCTTCTTCAAGATATTTTATTATAC

BB_D10 ACCTGCATATGAATTCAAAATTTATTTTAAAG

ATCAGCCCGGGACTGCCACCAAGTAATTTAAC

BB_E08 ACCTGCATATGCAAAAAAATGTTTATTG ATCAGCCCGGGTAAAACAAAATTTGATAAGCA

BB_E31 TGAAATACCATATAATCGTAAG ATCAGCCCGGGAAGGTTGCTTATTGACAA

BB_E04 ACCTGCATATGAAAAACCCCAAATCAA ATCAGCCCGGGATTTTGGTTATTTTCTGGA

BB_F20 ACCTGCATATGAACAAAAAATTTTCTATTTC ATCAGCCCGGGGTTGCTTTTGCAATATGAA

BB_F01 ACCTGCATATGAAATTATTAAAAATATTTATGTGTG

ATCAGCCCGGGACTTAAACCCTTTACACTT

BB_G25 ACCTGCATATGAAAAATTTAAAGACAAAAATT

ATCAGCCCGGGTCTTCTGCTGTATTTTTTG

BB_G01 ACCTGCATATGAGAAAAAGTTTGTTTTTAT ATCAGCCCGGGTTTTTTATTGAGACTTTCTAAAAC

BB_H18 GAGGAATGACATATGAAAATGAAGG TGAGCCCCCGGGTAGGCTAATACC BB_H32 ACCTGCATATGAAATATAATACGATTATAA

GC ATCAGCCCGGGAAGGGTATATATTAA

AATTATCTCG BB_H06 ACCTGCATATGAAAAAAAGTTTTTTATCAAT ATCAGCCCGGGTAATAAAGTTTGCTT

AATAGCT BB_H01 ACCTGCATATGAACAAATTATTGATATTCAT ATCAGCCCGGGGTAATAATGGTTGAA

CGTG BB_H37 ACCTGCATATGATTAAGGGAAAGGAGAG ATCAGCCCGGGAGACTTACTTAAAGC

ATTTTTTG BB_I28 ACCTGCATATGAAATGCCATATAATTGC ATCAGCCCGGGAATCCGACAGATCTG

G BB_I16 ACCTGCATATGAAATACCACATAATTACA ATCAGCCCGGGAAGTTTATATTTTGAC

ACTATAAG BB_I42 ACCTGCATATGAGGATTTTGGTTGG ATCAGCCCGGGTGTAGGTAAAATAGG

AACTG BB_I36 ACCTGCATATGAAAAACTTTAAATTAAATA

CTATT ATCAGCCCGGGATCAGCTTGATCTAG

TAGAT BB_I38 ACCTGCATATGAAAAACTTTAAATTAAATA

CTATT ATCAGCCCGGGATCAGCTTGATCTAG

TAGAT BB_I39 ACCTGCATATGAAAAACTTTAAATTAAATA

TTATTAA ATCAGCCCGGGATCAGCTTGGTTTAGT

AG BB_I14 ACCTGCATATGAAAAACAAAATAATTTTAT

G ATCAGCCCGGGGTTTTTTATTTTATCA

GCATG BB_I29 ACCTGCATATGAAAAACAACATAATTTTAT

G ATCAGCCCGGGATTCGGCAGTGATAA

TATG BB_K50 ACCTGCATATGAATTTAATAATTAAAGTGAT

GTTG ATCAGCCCGGGTCTAGAGTCCATATCT

TGC BB_K07 ACCTGCATATGAGTAAACTAATATTGGC ATCAGCCCGGGATTATTAAAGCACAA

ATGTATG

52

BB_K12 ACCTGCATATGAGTAAACTAATATTGGC ATCAGCCCGGGGCTTAAAGTTGTCAATGTT

BB_K48 ACCTGCATATGAATTTAATTAATAAATTATTTATTCTA

ATCAGCCCGGGTCTAGAGTCCATATCTTGC

BB_K19 ACCTGCATATGAAAAAATATATTATCAATTTAAGT

ATCAGCCCGGGATTGTTAGGTTTTTCTTTTCC

BB_K53 ACCTGCATATGAGGATTTTGGTTGG ATCAGCCCGGGTGTAGGTAAAATAGAAACTGG

BB_K52 ACCTGCATATGAAAAAGAACATATATATTTTG

ATCAGCCCGGGTTCATCAGTAAAAAGTTTTAATT

BB_K32 ACCTGCATATGAAAAAAGTTAAAAGTAAATATTTG

ATCAGCCCGGGGTACCAAACGCCATTCTTG

BB_K01 ACCTGCATATGAGAAAAAGTTTGTTTTTAT ATCAGCCCGGGTTTTTTATTGAGACTTTCTAAAAC

BB_J36 ACCTGCATATGAAAAGGAAAAGCAATATATG

ATCAGCCCGGGAAGCGCAACTTTTTTATAAAC

BB_J34 ACCTGCATATGATAAAAGGCAATACGT ATCAGCCCGGGTTTATCTTTATTTTTAGGCTTAAC

BB_J41 ACCTGCATATGAAAAACCTTAAATTAAATATTATT

ATCAGCCCGGGATCAGCTTGGTTTAGTAGAT

BB_J01 ACCTGCATATGAAATACCATATAATCGTAAG

ATCAGCCCGGGTCCTGCACTAGCTGTTTC

BB_J47 TGAGGAATATTAGCAATTGTATC ATCAGCCCGGGGTAAGAACTATTCTCCTTTTC

BB_Q46 ACCTGCATATGATTAAAAATGTAATTTATATTTTAC

ATCAGCCCGGGTCCTACAATCCAAATTTTG

BB_Q05 TGAAATACTATATATGTGTGT ATCAGCCCGGGAAGGTTACTTATTGAAAATATCTG

BB_Q03 ACCTGCATATGAGGATTTTGGTTGG ATCAGCCCGGGTAAAATTTTTCCATTAATTGTATTT

BB_Q47 ACCTGCATATGAATAAAAAAATGAAAATATTTATT

ATCAGCCCGGGCTGACTGTAACTGATGTATC

BB_Q35 ACCTGCATATGAAAATCATCAACATATTATT ATCAGCCCGGGGTTTTGCCAATTAGCTG

BB_Q89 ACCTGCATATGAACAAATTATTGATATTCAT ATCAGCCCGGGGTAATAATGGTTGAACGTG

N/A CCCAGGCTTTACACTTTATGC GGCCTCTTCGCTATTACGC

53

insert by PCR using Taq polymerase and a primer pair complementary to pSC:LP sequences 5’

of the NdeI site and 3’ of the XmaI site, respectively (pSCreen-fwd, 5’-

AAGGATTTGCCAAAGTCAG-3’; pSCreen-rev, 5’-GTAAAACGACGGCCAG-3’)(Figure 5

and Table 1). PCR-positive clones were sequenced to verify the expected insert sequence

(ACGT; Eton Bioscience). E. coli clones harboring the verified recombinant plasmids were

stored in LB containing 15% (v/v) glycerol at -80°C.

Next, B. burgdorferi B31-e2 were transformed with the verified plasmids by

electroporation (178), cloned by plating in semi-solid BSK-II and expanded in liquid BSK-II as

described (30). Cultures of putative transformants were screened by both PCR using gene-

specific primers (see Table 1) and by Western Blot of whole cell lysates with the HisProbe-HRP

reagent (Thermo Fisher) according to the manufacturer’s instructions. Clones that expressed

epitope-tagged protein were then expanded in BSK-II and used in subsequent assays. Verified B.

burgdorferi clones were stored in BSK-II containing 10% (v/v) DMSO at -80°C (177).

4.2.3 Surface proteolysis of intact B. burgdorferi spirochetes

Proteolytic shaving of intact spirochetes with proteinase K was performed as described (28-30,

180), with minor modifications. Treatment of cells with Pronase (Roche) followed previously

published protocols (186, 187) but used the manufacturer’s currently recommended reaction

conditions (Roche# 10165921001, Version 07). Briefly, cells were grown at 34˚C to late-

logarithmic phase in BSK-II and harvested by centrifugation at room temperature using a

centrifugal force not exceeding 3,000 x g using a Sorvall Legend RT centrifuge. Cells were then

washed once by resuspension in sterile, room temperature Dulbecco’s phosphate buffered saline

(dPBS) containing 5mM MgCl2 (dPBS+Mg) and re-pelleted. Cells were then resuspended in

54

dPBS+Mg with either dH2O (mock), proteinase K (Invitrogen, 200 µg/mL final concentration) or

Pronase (Roche Life Sciences, 1 mg/mL final concentration). Proteinase K-containing samples

and respective controls were incubated for 1 hour at room temperature, and reactions were

stopped after 1 hour by addition of PMSF to a final concentration of 5mM (188, 189). Pronase-

containing samples and respective controls were incubated for two hours at 37°C, and reactions

were stopped by addition of EDTA, PMSF, and Pefabloc SC to 1mM, 0.2mM, and 0.8mM final

concentrations, respectively. Subsequently, cells were pelleted by microcentrifugation,

resuspended in SDS-PAGE sample buffer containing 50mM dithiothreitol (DTT; final

concentration), boiled for 5 minutes and stored at -20°C until analysis by SDS-PAGE (190).

4.2.4 Isolation of B. burgdorferi OM Vesicles (OMVs)

OMVs from spirochetes were isolated as previously described (28-30, 181). Briefly, cells were

grown to early exponential phase, harvested, and washed with dPBS containing 0.1% (w/v)

bovine serum albumin. Cells were then resuspended in 25mM sodium citrate, pH 3.2 containing

0.1% (w/v) bovine serum albumin (BSA) (citrate buffer+BSA). Cell suspensions were shaken

for two hours at room temperature in a New Brunswick C24 incubator at 250 rpm to release

OMVs, at which point the cell suspension was harvested, resuspended in citrate buffer+BSA, and

loaded onto a discontinuous 56/42/25% (w/w) sucrose gradient in citrate buffer without BSA.

Cell suspensions were centrifuged at 100,000 x g for 18 hours at 4°C in a Beckman-Coulter

XPN-80 ultracentrifuge using a SW 32 Ti rotor and Beckman UltraClear tubes, and the resulting

upper (OMV) bands and lower (protoplasmic cylinder, PC) bands were separated by needle

aspiration. Fractions were diluted in cold dPBS, re-pelleted separately, and then resuspended in

dPBS containing 1mM PMSF. A portion of the resuspended fractions was used to prepare SDS-

55

Table 2. B. burgdorferi lipoproteome localization data. Localization data from the current study were reconciled with previously published data and genome-wide studies of in vivo gene expression, protein immunogenicity and requirement for in vitro growth. Column 1 lists the assayed lipoproteins by ORF (open reading frame) nomenclature (31, 32); ORFs that were identical in mature sequence to other analyzed ORFs are marked with an asterisk (*)(see Fig. 6 and text). Column 2 lists common protein names used in the literature. Column 3 states the determined consensus localization of the assayed lipoproteins, as described in the text. S, surface; P-OM, periplasmic-outer membrane; P-IM, periplasmic-inner membrane; nd, not detected. Column 4 lists the determined localization of the C-terminally His-tagged proteins (Figs. 6, 7 and 8). Localizations followed with a dot (•) indicate that the His-tagged protein was resistant to proteinase K (Fig. 6), but not pronase (Fig. 8). Column 5 lists the dNSAF ratio (dNSAF pK–/dNSAF pK+) determined by MudPIT analysis (see text). nd, not detected. ∞, infinite value due to lack of detection of any peptides after pK treatment, i.e., division by 0. Column 6 lists the previously determined and published lipoprotein localization along with the PubMed ID (PMID) of the associated publication. Columns 7 and 8 list the predicted and observed molecular masses (in kilodalton, kDa) of each protein. nd, not detected. Column 9 lists the paralogous family with key member and number according to Casjens et al. (31). Column 10 lists the observed in vivo expression pattern according to Iyer et al (19). Transcripts that showed significant elevation in the fed larval stage relative to at least one other stage were classified as important for tick acquisition (TA) or tick persistence (TP), as these genes were up-regulated in the transition from infected mice to naïve larvae. Transcripts that showed significant elevation in the fed nymph stage relative to at least one other stage were associated with vertebrate transmission (VT), based on their apparent importance for the spirochete’s passage from the feeding nymph to the naïve mouse. Finally, transcripts that were significantly elevated in dialysis membrane chambers (DMCs) relative to at least one other stage were considered necessary for vertebrate persistence(VP), given their induction in a quasi-steady state mammalian environment. Column 11 lists protein immunogenicity as determined by Barbour et al. (99). A Microsoft Excel version of this table is available upon request.

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58

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PAGE samples and stored at -20°C after boiling. The remainder of the sample was stored at -

80°C for later analysis.

4.2.5 SDS-PAGE analysis and immunoblotting

SDS-PAGE analysis and immunoblotting was performed as described (28-30). Protein

samples were prepared as described above and separated using a 12% SDS-polyacrylamide gel

(Bio-Rad). After transfer, gels were either Coomassie-stained for total protein (EZ-RUN Protein

Gel Staining Solution, Fisher) or were transferred to nitrocellulose membranes (Amersham, GE

Healthcare) using a Trans Blot SD apparatus (Bio-Rad). Membranes were then blocked post-

transfer with either 5% (w/v) nonfat dry milk or 2.5% (w/v) BSA and probed with mouse anti-

FlaB (H9724 (191); 1:300 dilution), mouse anti-OspA (H5332 (45); 1:1,000), rabbit polyclonal

anti-OppAIV ((34); 1:1,500) , or the HisProbe-HRP reagent according to the manufacturer’s

instructions. H9724 and H5332 antibodies were a gift from Dr. Alan Barbour (Univ. of

California at Irvine, CA), and anti-OppAIV antibody was generously provided by Dr. Patricia

Rosa (NIH/NIAID Rocky Mountain Laboratories, Hamilton, MT). Blots were treated with

corresponding AP-conjugated secondary antibodies (Sigma-Aldrich Cat. No’s A3562, A3687,

1:30,000) and developed using LumiPhos (Thermo Fisher, now discontinued) or Immun-Star AP

(Bio-Rad). Blots probed with HisProbe-HRP reagent were developed with SuperSignal West

Dura reagent (Thermo Fisher). Signals were detected and captured using a Fujifilm LAS-4000

CCD imager and further processed with Adobe Photoshop CS6.

4.2.6 Analysis of protein fractions by Multidimensional Protein Identification Technology

(MudPIT)

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Localization of lipoproteins endogenously expressed under standard culture conditions

was determined using multidimensional protein identification technology (MudPIT) mass

spectrometry (192). B. burgdorferi B31-A3 cells were subjected to surface proteolysis with

proteinase K as described above. Membrane-associated proteins were then enriched by

overnight extraction of the mock and proteinase K-treated samples with Triton X-114 as

described (186). The washed detergent extracts were then precipitated overnight using acetone

(80% v/v), resuspended in 0.1M Tris-HCl, pH 8.5, and precipitated again overnight using TCA

(20% v/v). The addition of acetone precipitation was in the protocol was necessary to effectively

remove detergent prior to analysis by MudPIT. Two biological replicates of the resulting

desiccated, frozen protein samples were then submitted for MudPIT analysis (Proteomics Center,

Stowers Institute for Medical Research; Kansas City, MO). Resuspended protein samples were

digested with endoproteinases Lys-C (Roche) and Trypsin (Promega) at 0.1 µg/µL final, each.

The protease-digested samples were then analyzed by MudPIT on an LTQ linear ion trap

(Thermo Scientific) coupled to a Quarternary Agilent 1100 series HPLC (193). Protein content

in mock control vs. proteinase K-treated whole cell protein preparations were analyzed by

comparison of the average dNSAF (distributed normalized spectral abundance factor) for each

unique protein, which correlates directly with the relative abundance of a particular protein in the

sample (194). A dNSAF ratio of control vs. protease-treated sample (dNSAF–pK/dNSAF+pK) was

calculated for each detected protein. Theoretically, a ratio of 1 indicates that the protein is as

abundant after surface proteolysis as before, i.e., not susceptible to proteinase K due to either

periplasmic localization or intrinsic resistance to protease. Conversely, a ratio greater than 1

indicates that a protein is less abundant after proteolytic shaving, i.e., surface exposed.

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4.3 Experimental Results

4.3.1 Generation of an epitope-tagged lipoprotein expression library in B. burgdorferi

Our long-standing interest in understanding the biogenesis of spirochetal envelopes,

particularly the sorting mechanisms for the numerous and abundant B. burgdorferi lipoproteins

(25, 28-30, 156, 174, 195, 196), has been continually thwarted by the quite limited and biased

data set of characterized lipoproteins. As shown in Table 2, 49 B. burgdorferi lipoproteins have

been localized independently to date, with over two thirds of them found on the bacterial surface.

We therefore set out to comprehensively localize the published list of proteins that are predicted

to make up the B. burgdorferi lipoproteome (23). This list was compiled by training a computer

algorithm (SpLip) on a set of spirochete proteins that had been experimentally verified to be

lipidated, and then using that trained algorithm to scan the published genome of B. burgdorferi

B31 (31, 32). A total of 127 putative open reading frames (ORFs) were annotated as “probable,”

“possible,” or “false negative” lipoproteins (23). These 127 ORFs were used as our “working

lipoproteome” for the creation of a C-terminally histidine-tagged expression library. This

approach allowed us to use a single commercial blotting reagent (HisProbe-HRP) to probe the

entire lipoprotein library, bypassing the need to generate individual and validated lipoprotein-

specific antibodies.

B. burgdorferi B31-e2 clones expressing each individual epitope-tagged lipoprotein were

obtained as described above. Of the 127 lipoproteins that were to be cloned, three (BB0536,

BB0652 (SecD), and BBQ46.) showed no expression of his-tagged protein in multiple B.

burgdorferi clones; all clones contained the respective recombinant plasmid when assayed by

PCR and DNA sequencing, indicating that the lack of expression was not due to absence of

plasmid or mutation of the promoter or coding sequence. These three ORFs were not pursued

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Figure 6. Surface proteolysis of B. burgdorferi strains expressing a his-tagged lipoprotein library using Proteinase K. Intact cells expressing the his-tagged lipoprotein were treated with Proteinase K or mock-treated as described in the text. Cell lysates were then separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western Blotting using anti-OspA or anti-FlaB mouse MAbs, or HisProbe-HRP reagent. Lipoproteins are organized according to open reading frame (ORF) nomenclature (31, 32), with the common name of the protein listed if applicable (Table 2). pK–, untreated mock control; pK+: proteinase K-treated sample. Red parentheses flanking the ORF designation indicate the determined lipoprotein localization as follows: ))ORF, surface; (ORF), periplasmic. A red dot (•) indicates proteins where the consensus localization was ultimately changed to the periplasm [•))] or surface [))•] due to independent data or follow-up pronase digestion (Figure 8A). Determined molecular weights of the his-tagged proteins are indicated in Table 2.

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further. In addition, four pairs of lipoproteins were found to be 100% identical in their mature,

processed sequences when analyzed by sequence alignment (T-COFFEE;

http://www.tcoffee.org/) (197): BBP38 (ErpA) and BBL39 (ErpN), BBP39 (ErpB) and BBL40

(ErpO), BBP27 (RevA) and BBM27 (RevA), as well as BBH01 and BBQ89. As it is all but

certain that proteins with identical primary sequences localize identically (25), each of these

pairs is represented by a single member (the first ORF listed of the pair). This resulted in an

assayed dataset of 120 unique lipoproteins covering 124 members (or 98%) of the predicted B.

burgdorferi lipoproteome.

4.3.2 Initial assignment of lipoproteins to the bacterial surface based on proteinase K

accessibility

We used an established and validated stepwise experimental protocol to individually

localize each epitope-tagged lipoprotein within the spirochetal cell envelope. As described, we

first subjected each recombinant B. burgdorferi clone to in situ surface proteolysis (or

“proteolytic shaving”) with proteinase K, a membrane impermeable, nonspecific protease that

selectively digests surface lipoproteins in the context of an intact OM (28-30). Surface

lipoprotein OspA was used as a positive control as it is readily degraded by proteinase K under

the assay conditions. Conversely, the periplasmic flagellar protein FlaB was used as a negative

control to ascertain OM integrity of the assayed cells. The His-tag epitope was used to assess the

sensitivity of the tagged lipoprotein to Proteinase K, assuming that the localization of the C-

terminal his-tag mirrors that of the lipoprotein itself. Of note, C-terminal processing of secreted

proteins in B. burgdorferi is rather specific and limited to a small set of proteins (198, 199), and

C-terminal tags are not known to alter lipoprotein localization (29, 200). Lipoproteins that lost

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the his-tag signal upon proteolysis were considered to be surface-exposed (S), whereas

lipoproteins that showed no loss in signal relative to the controls were considered to localize to

the periplasmic (P) face of the OM (OM) or inner membrane (IM). Compiled Western blot

results for each of the assayed lipoproteins are shown in Figure 6, organized by ascending ORF

nomenclature. Of the 124 lipoproteins covered by the analysis, were classified as surface-

exposed, while 41 were considered to localize to the periplasm. Interestingly, a subset of

lipoproteins exemplified by the Mlp protein family showed only partial degradation of the his-

tag after proteinase K treatment (Figure 6). This could indicate that only fractions of these

lipoproteins are exported to the surface. Alternatively, it could reflect the lipoproteins’ native

folding, which may render their C termini less accessible to protease in the context of the

spirochetal envelope.

4.3.3 Initial assignment of periplasmic lipoproteins to the outer or inner membrane by

membrane fractionation

Recombinant B. burgdorferi clones that expressed epitope-tagged lipoproteins protected

from surface proteolysis with proteinase K were subjected to membrane fractionation. OM

vesicles (OMVs) and protoplasmic cylinder (PCs) fractions were obtained as described above by

incubating harvested cells in a hypotonic citrate buffer followed by loading on a step-wise

sucrose gradient. Note that due to the not entirely efficient separation of the OM during the

process (181), the PC fraction should be interpreted as a partially OM-depleted whole cell

protein fraction. Thus, the surface/OM control OspA is abundant in the OMV fractions, but also

detected in the PC fractions (29, 30). In contrast, the inner membrane lipoprotein OppAIV can

be used as a control to assess the purity of the OMV preparation, as it should be absent from an

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ideal OMV preparation. A lipoprotein was scored as an OM component if the His-tag was

detected in the OMV fraction in a ratio similar to the OspA control. Absence or only traces of a

His-tag signal from the OMV fraction indicated that the lipoprotein was retained in the inner

membrane. As shown in the Western immunoblots in Fig. 7, 31 of the 40 lipoproteins assayed

showed an OppAIV-like fractionation pattern, i.e., they were retained in the IM. Conversely, 9

lipoproteins were detected in appreciable amounts in the OMV fraction, indicating that they were

released to the OM.

4.3.4 Reassessment of select OM lipoproteins for potential intrinsic proteinase K resistance

The lipoprotein BBQ47 (ErpX) was previously established as a surface-exposed but

intrinsically proteinase K resistant protein (187). Consequently, the ErpX-expressing B.

burgdorferi clone was not subjected to membrane fractionation despite the protein’s apparent

resistance to proteinase K. Yet, this example raised the specter that other surface lipoproteins

may have been erroneously scored as OM periplasmic lipoproteins due to their resistance to

proteinase K. We therefore re-evaluated the 9 lipoproteins in our initial P-OM lipoprotein data

set using pronase, a mixture of nonspecific proteases that has been shown to digest B.

burgdorferi proteins that are otherwise protease-resistant (186, 187). ErpX was used as a

control.

As shown in Figure 8A, pronase treatment led to complete degradation of OspA, while

FlaB remained intact, indicating that assay conditions lead to selective removal of surface-

exposed proteins. Parallel treatment of B. burgdorferi B31-A3 cells and staining of SDS-PAGE-

separated protein samples by Coomassie (Figure 8B) indicated almost indistinguishable overall

proteolysis patterns between pK and pronase; the only appreciable difference in the pronase-

68

treated sample was the absence of a 51 kDa band that has been attributed to a proteinase K-

resistant fragment of the OM porin P66 (201). As expected, the control protein BBQ47 (ErpX)

was largely susceptible to degradation by pronase. Three additional lipoproteins, BBA14,

BBA65, and BBB25, showed selective degradation by pronase as well. The remaining 6

lipoproteins were found to be pronase-resistant in the context of intact cells (Fig. 8A), but readily

digested and undetectable when the cells were permeabilized (not shown). This indicated that

these 6 proteins are not intrinsically protease-resistant, but indeed localize to the periplasmic

leaflet of the OM. In summary, this set of experiments localized BBA14 and BBB25 to the

surface de novo. BBA65 had been localized previously (202). In that study, the authors found

that BBA65 was susceptible to both pronase and proteinase K; the latter could be due to the

higher concentration of proteinase K used (400 µg/mL). It also established BB0460, BB0475,

BBB09, and BBG25 as additional bona fide P-OM lipoproteins and confirmed the previous

localization results for both BB0324 and Lp6.6 (87, 203).

4.3.5 Localization of endogenously expressed lipoproteins by quantitative mass spectrometry

To validate our lipoproteome expression library data with the localization of lipoproteins

endogenously expressed by B. burgdorferi, we employed quantitative MudPIT mass

spectrometry to analyse the lipoproteome of B. burgdorferi B31-A3 (184, 193). Our stock of

B31-A3 was shown by multiplex PCR (185) to contain all linear and circular plasmids except for

lp5, which is not predicted to encode for any lipoproteins (23) (data not shown). Cells were

cultured and treated with proteinase K as above. To reduce the complexity of the samples, the

mock control and proteinase K-treated samples were enriched for membrane-associated proteins

by extraction with Triton X-114 as described (186). Peptide abundance, expressed as distributed

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Figure 7. Membrane fractionation of B. burgdorferi strains expressing a his-tagged lipoprotein library. Strains that were found to express proteinase K-resistant recombinant lipoproteins were subjected to membrane fractionation using a hypotonic acidic citrate buffer and sucrose gradient to obtain OMVs. Lipoproteins were then localized based on presence or absence in OMV fraction relative to control proteins. OMVs and PCs were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western Blot using anti-OspA mouse MAb, anti-OppAIV rabbit polyclonal antiserum, or HisProbe-HRP reagent. Lipoproteins are organized by open reading frame with the common name listed, if applicable. OMV, outer membrane vesicle fraction; PC, protoplasmic cylinder fraction. Note that the PC fraction is equivalent of a whole cell protein preparation partially depleted of OM proteins (see text). Red parenthesis flanking the ORF designation indicate the determined lipoprotein localization as follows: (ORF, inner membrane; ORF), outer membrane. The localization of BBA65 remains undetermined [(ORF)] due to multiple isoforms with variable distributions. Determined molecular weights of the his-tagged proteins are indicated in Table 2.

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Normalized Spectral Abundance Factor (dNSAF) (194), was captured from two biological

replicates and averaged. A comparison of the MudPIT results with the results from our his-

tagged lipoprotein assays can be seen in Table 2 and Fig. 9. 86 of the predicted 127 lipoproteins

were detectable in B. burgdorferi B31-A3 after growth at 34°C in BSKII-C. Among them were

2 of the 3 lipoproteins that were not detectable as His-tagged proteins, BB0536 and BB652

(SecD). The remaining 40 lipoproteins are most likely expressed below the levels detectable by

MudPIT under the culture conditions used.

The relative abundance of proteins before and after proteolytic shaving, expressed as a

ratio of dNSAF values in the mock control vs. the treated samples (dNSAF-pK/dNSAF+pK), was

calculated, ranging from 0.00 (BB0628) to 185.84 (BBA16/OspB). 11 lipoproteins were

undetectable after proteolysis, resulting in an infinite (∞) ratio; for analysis purposes, the values

of these proteins were capped at the highest calculated value (185.84). Plotting the dNSAF

ratios for the surface and periplasmic lipoprotein cohorts identified in the expression library

showed a clear separation (Fig. 9). The mean dNSAF ratio for periplasmic lipoproteins was

0.80(range: 0.00-2.22), below but close to the expected ratio of 1.0. One explanation is that

surface proteolysis yields a significantly less complex protein sample (see Fig. 8B), which may

lead to the “unmasking” of previously undetectable/unassignable peptides. Consequently, the

ratio’s numerator may be depressed relative to the denominator. The mean dNSAF ratio for

surface proteins was 60.00 (range: 0.00-185.84 [capped; see above]). 12 surface-assigned

lipoproteins had low dNSAF ratios around or below 1.0. Most of the proteins in this cohort were

members of the paralogous Mlp, Rev, and Erp protein families that had shown at least partial

resistance to proteinase K in our experiments (Figure 6) or prior studies (Table 2). Also, 4 of

these proteins were subsequently shown to be accessible to in situ pronase digestion (Fig. 8A).

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As shown in Fig. 9, a dNSAF ratio of 2 or above is a high-confidence predictor for surface

exposure. This cutoff was statistically confirmed by a Mann-Whitney non-parametric U test

using the OriginPro 9.1 software suite. Parallel analysis of sequence coverage, i.e., the % of

protein sequence covered by detected peptides, before and after pK treatment also showed that a

reduction of 10% or higher was a statistically valid predictor of surface exposure (see Fig. S1

and Table S1).

All mass spectrometry data are available from the ProteomeXchange repository under

accession number PXD005617. The complete MudPIT mass spectrometry dataset (raw files,

peak files, search files, as well as DTASelect result files) can be obtained from the MassIVE

database via ftp://MSV000080434@massive.ucsd.edu (password ASD06166).

4.3.6 Reconciliation of independent localization data produces a consensus localization catalog

for the B. burgdorferi lipoproteome

Our lipoproteome expression library contained 49 lipoproteins that had been

independently localized prior to this study (Table 2). The main rationale for their inclusion in

the present study was to use them as internal validation controls. For 41 lipoproteins, the current

localization data were in unequivocal agreement with published data. Three lipoproteins,

BB0298, BBA05/S1 antigen and BBA34/OppA5, were more precisely localized within the

periplasmic compartment to the IM. Discrepancies with previously published localization data

were found for 8 lipoproteins, but all disagreements could be reconciled: (i) A first set of 4

proteins was previously described as surface-exposed but was found to be restricted to the

periplasm in our assays (see Table 2). BmpB (BB0382), like the other homologs of this protein

family, was localized by us to the IM. An earlier study had detected BmpB on the surface by

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Figure 8. Surface proteolysis of B. burgdorferi strains expressing a his-tagged lipoprotein library using Pronase. (A) Intact cells expressing his-tagged lipoproteins that were determined to be Proteinase K resistant but enriched in the OM were subjected to surface proteolysis with Pronase. Cell lysates were then separated by SDS-PAGE and analyzed by Western Blot as in Fig. 6. Pronase –, untreated mock control; Pronase +: Pronase-treated sample. Red parentheses flanking the ORF designation indicate the determined lipoprotein localization as in Fig. 6: ))ORF, surface; (ORF), periplasmic. (B) Coomassie-stained SDS-PAGE gel of B. burgdorferi strain B31-A3 whole cell protein preparations obtained before (–) or after (+) incubation with proteinase K or Pronase. Protein MWs in kDa, indicated to the left, were derived from a protein molecular weight marker (BioRad). An asterisk (*) indicates a known proteinase K-resistant, but apparently Pronase-sensitive fragment of integral OM protein P66 (201).

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immunofluorescence, albeit without controlling for accidental permeabilization of the fragile

spirochetal OM (204). OppA1 and OppA2 were also localized to the IM, as were the other

homologs of that oligopeptide-binding lipoprotein family. Again, prior studies had solely relied

on immunofluorescence data without controlling for the integrity of the bacterial envelope (205,

206). The fourth protein in this set, BBA03, was also found solely in the IM in the present study.

A prior well-controlled localization study remained equivocal in that BBA03 was found by

immunofluorescence to be partially surface-exposed, but at the same time was protected from

proteinase K in intact, but not permeabilized cells (207). (ii) Two IM lipoproteins, BB0227 and

BBB27, were localized to the inner leaflet of the OM in one of our earlier studies (29); we now

believe that these localization results were erroneous due to contamination of the OMV fraction

and a less stringent interpretation of the data. (iii) BB0323 was localized by us to the IM but was

identified by others as an OM periplasmic lipoprotein (208). BB0323 was shown to undergo

multistep proteolytic processing in the periplasm (209). Consequently, upon protein maturation,

any C-terminal epitope tag will partition together with a C-terminal soluble fragment.

Accordingly, we detected only a 14-kDa fragment of the 44-kDa full-length protein in the PC

fraction (Table 2). We therefore defer to the previous work on BB0323 as a more accurate

reflection of this lipoprotein’s localization. (iv) Our final, but probably most intriguing

disagreement with a previous finding was BB0028. This lipoprotein was found to be at least

partially surface-exposed in our work but has been previously described as a periplasmic OM

associated with the OM Beta-barrel Assembly Machinery (BAM) complex (203). In other

bacterial systems, BAM lipoprotein modules were shown to assume topologies that expose their

C termini on the bacterial surface under certain experimental conditions (125, 210). Thus, we

hypothesize that BB0028 transiently and partially (i.e., predominantly via its C terminus)

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localizes to the surface. Any excess of BB0028 in the B. burgdorferi BAM complex may

detectably shift the protein’s topological equilibrium towards the cell surface.

The aggregation of the present and past data produces a comprehensive and internally validated

consensus catalog of lipoprotein localization within the B. burgdorferi envelope (Table 2). Of

the spirochete’s 127 predicted lipoproteins within this set, 125 localize conclusively to either the

surface (86), periplasmic leaflet of the OM (8), or the IM (31). The remaining two predicted

lipoproteins remained undetectable as epitope-tagged proteins and could not be conclusively

localized, but one (BB0536) was detected by mass spectrometry with a dNSAF ratio consistent

with periplasmic localization.

4.3.7 Reassessment of potential B. burgdorferi lipoprotein sorting motifs within the N-terminal

tethers

Our earlier studies indicated that lipoprotein tether peptides are structurally similar in that

they are intrinsically disordered. Yet, they lack any significant peptide sequence homology

beyond the N-terminal cysteine residue. Together with our extensive mutational analyses, this

led us to conclude that there are no specific canonical peptide motifs that direct lipoproteins to

the different envelope compartments of B. burgdorferi (29). With the lipoproteome localization

data in hand, we decided to reopen this inquiry. Tether peptide sequences from the cohorts of

surface, periplasmic OM and periplasmic IM lipoproteins were aligned and compared. Again, no

compartment-specific peptide motifs emerged from this analysis (Fig. 6). A recent study of

lipoprotein secretion in the Gram-negative pathogen Capnocytophaga canimorsus showed that

N-terminal patches of negatively charged Asp and Glu residues in the proper sequential and

positional N-terminal context can drive lipoprotein surface localization, and that this surface

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Figure 9. Scatter plot of MudPIT-derived dNSAF ratios of surface and periplasmic lipoproteins. dNSAF ratios before and after proteinase K treatment (dNSAF-pK/dNSAF+pK) were calculated for 87 lipoproteins detected by MudPIT and plotted using GraphPad Prism. Horizontal lines indicate the mean dNSAF ratios for surface and subsurface proteins. Note that (i) infinite dNSAF values due to undetectable protein after pK treatment were capped at the highest calculated value of 185.84 for BBA16/OspB, and (ii) surface-assigned proteins that are partially resistant to pK (see Fig. 6 and text) cluster with subsurface proteins (see Table 2 for specific dNSAF values).

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lipoprotein secretion signal is conserved and recognized in other members of the phylum

Bacteroidetes (212, 213). While we cannot fully exclude a similar charge-based sorting

mechanism in Borrelia barring additional experimentation, we did not detect any positional

conservation of negative charges in the 86 B. burgdorferi surface lipoproteins (Fig. 10).

4.4 Discussion of Experimental Results

4.4.1 Overview of results

The lipoprotein repertoire of B. burgdorferi is exceptionally large, especially when taking

into account the spirochete’s small and fragmented genome. Compared to E. coli’s 4-Mb

circular genome with about 90 lipoprotein genes (214, 215), B. burgdorferi’s 1.6 Mb genome

spread across a linear chromosome and a collection of linear and circular plasmids encodes for

over 120 lipoproteins. Since the initial identification of OspA and OspB as a common surface

antigens of Lyme disease spirochetes (45, 216) three decades ago, numerous studies have shown

that lipoproteins play an outsized and multifunctional role as virulence factors in the

transmission, colonization, dissemination and persistence of Borrelia burgdorferi and the

resulting pathology of Lyme disease. Following OspA’s and OspB’s precedent, much of the

research focus has been on identifying additional surface lipoproteins that contribute to B.

burgdorferi’s interface with the tick vector or host and could serve as vaccine targets. Thus, it

may be not surprising that 36 of the 47 additional lipoproteins that were subsequently studied

localized to the surface. Together, these studies yielded a rather limited and potentially biased

data set, covering less than 40% of the predicted B. burgdorferi lipoproteome. In the present

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study, we have closed this gap in our understanding of the complex cell envelope of B.

burgdorferi by providing spatial information on 98% of its predicted lipoproteome.

4.4.2 Correlation of gene and protein localization

It has been noted earlier that B. burgdorferi’s megabase linear chromosome, and to some

degree the cp26 “minichromosome”, tend to encode for essential genes, while the remainder of

the circular and linear plasmids appear non-essential for growth in culture (31, 37, 217-219).

While this generalization at least partially extends to the lipoproteins encoded by these replicons,

there is a remarkable correlation of gene localization and protein localization: 79 of the 90

plasmid-encoded lipoproteins (88%) are surface-exposed, while only 7 out of the 37

chromosomally encoded lipoproteins (19%) are found on the surface (Table 2). This dichotomy

is understandable when considering the biological function and primary protein sequence. Many

of the plasmid-encoded lipoproteins share significant homology, which has led to their

organization into “paralogous gene families” (31). Instructive examples are the Erp, Mlp, and

“Pfam54” lipoproteins encoded on the multiple prophage-related B. burgdorferi cp32/lp56

plasmids (31, 100, 183, 220). Overall, 62 of the 86 surface lipoproteins (72%) belong to a

paralogous group. By contrast, only 14 of the 39 periplasmic lipoproteins (36%) have other

paralogs (31) (Table 2). This well-titrated variability and redundancy in surface lipoproteins

may stem from the need of B. burgdorferi to adapt to a multitude of environments during its

enzootic cycle. Chromosomally encoded but surface-localized BB0352 and BB0689 were

identified to possess putative sugar- and lipid-binding domains by the HHPRED algorithm (221).

This suggests that surface lipoproteins encoded by essential genetic elements play housekeeping

or metabolic roles. Similarly, sub-surface lipoproteins located on non-essential plasmids seem to

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Figure 10. Sequence alignment of B. burgdorferi surface, periplasmic OM or IM lipoprotein tether peptides. A LogoBar (211) representation of the N-terminal sequence of known or predicted mature B. burgdorferi lipoproteins (23) illustrates the maintained complexity of surface (S), periplasmic OM (P-OM) and IM (P-IM) lipoprotein tether peptides. The height of each column, measured in bits, is proportional to the lack of complexity at a given position. The columns are stacked from the bottom starting with the most frequently occurring residue at that position and continuing upward. Below each column are the six most frequently occurring residues at each position, in order of frequency from top (bold) to bottom. Colors represent residues with similar characteristics (e.g., red for negatively charged Asp and Glu residues).

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have a role in transmission and virulence. BBJ47, which we localized to the IM, was identified

by HHPRED to belong to Pfam17044. This groups BBJ47 with the B. burgdorferi BptA

proteins and suggests a role in tick persistence (222).

4.4.3 Correlation of lipoprotein localization to in vivo expression and immunogenicity

To gain further insight into the biological significance of our data, we explored potential

correlations of lipoprotein spatial compartmentalization with temporal expression and

immunogenicity (Table 2). Our working lipoproteome data were first cross-referenced with a

previous study that examined the reactivity of Lyme borreliosis patient vs. control sera to various

B. burgdorferi proteins (99). 43 of the 127 lipoproteins were found in that study to be

immunogenic (Table 2). Next, we referenced our data set against a study that looked at the

transcription of B. burgdorferi genes at various stages of the spirochete’s enzootic cycle, using

an RNA-hybridized microarray assay that was validated through qRT-PCR (19). 53 lipoproteins,

mostly plasmid-encoded and surface-localized, were found to have some variable role in the

infectious cycle based on significantly different levels of transcripts during tick acquisition, tick

persistence, vertebrate transmission and vertebrate persistence (Table 2). Of note, this data

mining approach neglects any proteins that may be essential to the cell but are not differentially

regulated between environments. Immunogenic surface lipoproteins expressed during tick

acquisition, tick persistence or vertebrate transmission may work in a preventive setting,

similarly to the FDA-approved but no longer available OspA vaccine (109). Within this group

are BBA04 (S2 antigen), BBA07, BBA16 (OspB), BBB19 (OspC), BBK53, and BBL40 (ErpO).

Both OspB and OspC have previously been shown to elicit protection when used to immunize

laboratory mice (223, 224). Of the remaining four lipoproteins (BBA04, BBA07, BBK53, and

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BBL40), BBA07 has been implicated in transmission from the tick to vertebrate host and BBL40

is part of the Erp protein family, a group of lipoproteins implicated in Factor H binding and

complement resistance (72, 225).

4.4.4 Mechanistic insights into tether peptide-mediated lipoprotein sorting in diderm bacteria

Our results show that two thirds of the lipoproteins expressed by B. burgdorferi are

exported to the cell surface, while the remaining periplasmic lipoproteins are mostly retained in

the inner membrane. Whereas the periplasmic OM lipoprotein Lp6.6 is among the most

abundant envelope proteins (88), the relative simplicity of the B. burgdorferi lipoproteome in the

inner leaflet of the OM is unexpected. In E. coli, an organism with a diderm membrane

architecture similar to that of B. burgdorferi, most of the lipoproteins are exported to the OM as

well (226), but the majority are not surface exposed (126). A generalization that lipoprotein

surface exposure is rare and limited appears to hold for most diderm bacteria, with the emerging

exception of the Gram-negative Bacteroidetes such as Bacteroides fragilis and C.

capnocytophaga (212, 213, 227). Yet, the identified charge-based C. capnocytophaga

lipoprotein secretion signals appear to be restricted to that phylum (212, 213), and the B.

burgdorferi surface localization determinants identified in our own studies appear to extend only

to other members of that expanding genus (196). This hints at significant mechanistic

differences in lipoprotein secretion between Gram-negative bacteria and spirochetes. Our

current data remain compatible with a lipoprotein secretion model that includes two separate

secretion checkpoints: The first checkpoint is set up the IM where some lipoproteins are retained

and others are released to the OM after completion of N-terminal processing. Whether

lipoprotein retention and release is generally mediated by the partial B. burgdorferi Lol pathway

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Table 3. Comparison of lipoprotein tether length based on localization. Lipoprotein tethers for each localization phenotype were either predicted in silico using PSSPRED (228) or using crystal structure PDB data, if available. PSSPRED predictions were performed using the mature lipoprotein sequences from the residue immediately following the acylated cysteine. Lipoproteins are annotated using the standard B. burgdorferi genetic locus notation (31, 32), with the PDB structure given in parentheses if available. Lipoprotein localizations were taken from consensus localization data (see Table 2). Representative lipoproteins were chosen so that only a single member of each paralogous family (Table 2) was represented. Ellipses and numbers indicate the number of tether residues omitted for clarity.

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+1 +2 +3 +4 +5 +6 +7 +8 +9 +10 Surface

BBA15 (3AUM) C K Q N V S S L D …(3) BBA24 (4ONR) C G L T BBA68 (5A2U) C A P F S K I D P …(40)

OM-PERI

BBA62 C E T T R I BB0028 C S S BB0324 C Y T I N L E K L …(5)

IM

BBB16 (4GL8) C V N E S N R N K …(1) BB0215 (4N13) C K N Q D N E

BB0141 (4KKT) C V G D N K L D D …(12)

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(25) and dependent on specific N-terminal tether peptide residues is under current investigation.

Alternatively, release from the IM may be hindered by functional interactions of folded IM

lipoproteins with other IM protein complexes. Multiple lines of evidence point to a similar

“exclusion” mechanism at the second checkpoint in the OM, where periplasmic lipoproteins are

blocked from “flipping” through the OM due to assumption of their final tertiary structure (28-

30, 174, 195). The B. burgdorferi OM β-barrel assembly machinery (BAM) protein BamA was

shown to be at least indirectly involved in this process (229), and it remains to be determined

if/how the other identified B. burgdorferi BAM complex proteins (203, 210), including the IM

protein TamB (230), play a role in lipoprotein secretion. Recent work has shown that some

Neisseria surface lipoproteins are secreted to the surface through the lumen of β-barrel integral

OM proteins (172), but homologs are missing from Borrelia genomes. Together, this supports

our earlier notions that lipoprotein secretion pathways in Borrelia spirochetes are unique.

4.4.5 Final Conclusions Regarding Localization of the B. burgdorferi Lipoproteome

We have comprehensively localized the B. burgdorferi lipoproteome using an epitope-

tagged expression library and validated our findings by proteomics and by reconciling our data

with prior independent protein localization data. The used approaches, such as the initial

reliance on C-terminal epitope tags for protein detection, are not without limitations that will

only be eliminated with the generation of protein-specific antibodies and further in-depth studies

of the newly localized proteins. Yet, we are confident that the consensus B. burgdorferi

lipoproteome localization catalog presented here reflects the proteins’ native partition in the

spirochetal cell envelope. Lyme borreliosis remains the most common vector-borne illness in the

United States and is common throughout temperate climates in the Northern hemisphere, and

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there are continuing efforts to improve diagnostics and preventive measures. Placing our

lipoproteome localization data into the context of genome- and proteome-wide studies may

facilitate the identification of additional diagnostic targets and vaccine candidates. The

proteomic localization data presented here also provide a predictive framework for proteomic

studies of host-pathogen interfaces and envelope structures of other members of the ever

expanding Borrelia genus, including the relapsing fever spirochete Borrelia miyamotoi (231,

232) and the recently described North American Lyme disease spirochete Borrelia mayonii (233,

234).

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Chapter 5: Detailed Experimental Protocols

5.1 Preparation of basal Barbour-Stoenner-Kelly II (BSK-II) Medium (175)

1. Pre-rinse all glassware to be used thoroughly with Milli-Q ultra-pure dH2O.

2. Prepare 1L of 1X CMRL-1066 w/o l-glutamine (U.S. Biologicals# C5900-01) in a 2L

glass beaker by adding 9.7g of powder to approximately 1L of Milli-Q dH2O. Gently

mix using a magnetic stir bar.

3. Dissolve 5g of neopeptone (BD Bacto# 211681) into solution.

4. Dissolve 50g of Probumin bovine serum albumin, universal grade (EMD Millipore#

810037) slowly into solution using a magnetic stir bar. This process should take at least

an hour at room temperature.

5. Dissolve the following reagents into solution in order, making sure that each has

dissolved before adding the next. Where no manufacturer is indicated, use ACS grade

reagent.

a. 2g of TC Yeastolate (BD Bacto# 255772)

b. 6g of HEPES, free acid

c. 5g of dextrose (D-glucose)

d. 0.7g of sodium citrate (tribasic dihydrate)

e. 0.8g of sodium pyruvate

f. 0.4g of N-acetyl-D-glucosamine

g. 2.2g of sodium bicarbonate

6. Adjust the pH of the medium to 7.6 using 1M NaOH. The NaOH solution should be no

more than one week old.

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7. Filter sterilize the solution using two 0.22µm PES filter units (Corning# 431097) using

fiberglass prefilters (Corning# 431412). Prefilters should be pre-wetted using Milli-Q

dH2O and applied to the top of the filter unit under vacuum before applying medium.

8. Under a biological safety hood, aseptically remove the vacuum unit from the bottles and

cap. Distribute the medium evenly between the two bottles so that each contains

approximately one-half of the total preparation of medium.

9. Store up to six months in the dark at 4°C.

5.2 Preparation of complete BSK-II Medium (175)

1. Equilibrate one bottle of basal medium (see Section 5.1) to 37°C in a water bath.

2. Prepare a 100mL solution of 7% (w/v) gelatin:

I. Pre-rinse a 250mL bottle thoroughly with Milli-Q dH2O.

II. Measure 7g of gelatin (BD Difco# 214340) into the bottle.

III. Add approximately 95mL of Milli-Q dH2O to the bottle, swirl gently to mix, and

incubate at room temperature 10 minutes to hydrate the gelatin.

IV. Solubilize the gelatin, either using a microwave or by autoclaving.

V. Equilibrate to 37°C in a water bath.

3. Equilibrate a 40mL aliquot of sterile, trace-hemolyzed rabbit serum (Pel-Freez# 31126-5)

to 37°C in a water bath.

4. Thoroughly rinse a 1L bottle with Milli-Q dH2O. Combine the basal BSK-II, gelatin, and

rabbit serum in the 1L bottle and swirl to mix. (Note: At this stage, a portion of medium

can be measured out using a pre-rinsed glass graduated cylinder for the addition of

additives, e.g. antibiotics).

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5. Filter sterilize the medium using 0.22µm PES filter units (Corning# 431096 & 431097).

This step must be completed before the medium has cooled to below room temperature;

otherwise, the gelatin will begin to solidify and impede filtration of the medium.

6. Aseptically cap the bottles under a biological safety hood. Store the completed medium

at 4°C in the dark and should be used within one month of preparation for optimal results.

Before use, medium must be equilibrated to 34-37°C due to solidification of gelatin at

lower temperatures.

5.3 Preparation of Competent B. burgdorferi (177, 178)

1. Inoculate 500mL of complete BSK-II with 1mL of the desired culture in late-logarithmic

growth phase.

2. Incubate culture at 34°C until the cell density reaches approximately 5x107 cells/mL.

3. Divide the culture evenly between two sterile 500mL centrifuge bottles. Manipulation of

B. burgdorferi in this step, as well as in all following steps, must be performed aseptically

under a biological safety hood.

4. Pellet cells at 4,000 x g for 20min at room temperature. Carefully decant the supernatant.

5. Resuspend each cell pellet in 30mL of sterile, ice-cold phosphate buffered saline (pH 7.4)

+ 5mM MgCl2. Transfer cell suspensions to two sterile, conical 50mL tubes (Corning#

352098) that had been pre-chilled on ice. Keep cells cold or on ice from this point

forward.

6. Re-pellet cells at 3,000 x g for 15min at 4°C. Carefully remove the supernatant with a

serological pipette.

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7. Resuspend each cell pellet in 10mL of sterile, ice-cold 272mM sucrose. Transfer cell

suspensions to two sterile, conical 15mL tubes (Corning# 352097) that had been pre-

chilled on ice.

8. Re-pellet cells at 2,000 x g for 10min at 4°C. Carefully remove the supernatant using a

serological pipette.

9. Repeat Steps 7 and 8, but without transferring the cell suspensions to new tubes.

10. Pool the cell pellets by resuspension in 1mL of sterile, ice-cold 272mM sucrose. Cells

should be resuspended completely – no particulates should be visible when pipetted.

Divide cell suspension into 50µL aliquots into sterile, pre-chilled 1.5mL microcentrifuge

tubes on ice for electroporation. Alternatively, cells can be resuspended in 1mL of

sterile, ice-cold 272mM sucrose + 15% (v/v) glycerol, divided into aliquots, and flash-

frozen at -80°C for long-term storage.

5.4 Electroporation of B. burgdorferi (177, 178)

1. Thaw a 50µL aliquot of competent cells on ice for 5min, if using a pre-made stock.

2. Add 1-5µL of DNA in dH2O to competent cells and flick gently to mix. The amount of

DNA needed varies depending on the genetic background of the competent cells and on

the nature of the DNA to be transformed. High-passage B. burgdorferi B31 can be

transformed with as little as a few hundred nanograms of the shuttle vector pBSV2,

whereas low-passage (infectious) B. burgdorferi strains require approximately 30µg total

DNA. Preparations of DNA should be free from salts to minimize risk of arcing during

electroporation. Manipulation of cells in this step, as well in all following steps, must be

performed aseptically.

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3. Incubate cells and DNA on ice for 1min.

4. Transfer cells/DNA to a sterile 0.2cm electroporation cuvette (Bio-Rad# 1652086) that

had been pre-chilled to 4°C. Tap the cuvette gently to bring the cells/DNA liquid to the

bottom of the chamber, so that the liquid spans the void between the two electrodes.

5. Electroporate the cells using a single exponential decay pulse on a Bio-Rad GenePulser at

2.5kV, 25µF, 200Ω. Alternatively, a Bio-Rad MicroPulser can be used on the “Ec2”

setting and should optimally result in a time constant of approximately 5.8ms.

6. Immediately (within 1 minute of electroporation), add approximately 1mL of pre-

warmed (34°C) complete BSK-II to the cells in the cuvette using a sterile Pasteur pipette

(Fisher# 13-711-20). Pipette to mix, then transfer the cells to a 15mL Falcon tube

containing an additional 11mL of pre-warmed complete BSK-II. Cap the tube and invert

several times to mix. At this stage, antibiotics should not be used unless the genetic

background of the competent cells requires it.

7. Incubate at 34°C for 18-24 hours to allow for outgrowth of transformants.

5.5 Plating and Selection of B. burgdorferi Transformants (177, 178)

1. Prepare 2X basal BSK-II medium using the protocol as described in Section 5.1,

except that the initial volume of dH2O is reduced by half.

2. Measure 250mL of 2X basal BSK-II using a pre-rinsed glass graduated cylinder.

Dissolve necessary antibiotics at 2X necessary concentration.

3. Filter-sterilize 250mL of 2X basal BSK-II+antibiotics and 30mL of trace-hemolyzed

rabbit serum using a 500mL 0.22µm filter unit. Aspetically cap bottle and equilibrate

to 50°C in a water bath.

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4. Prepare a 220mL solution of 1.52% (w/v) agarose:

i. Pre-rinse a 500mL bottle thoroughly with Milli-Q dH2O.

ii. Measure 3.33g of SeaKem LE agarose into bottle.

iii. Add approximately 220mL of Milli-Q dH2O. Microwave until agarose

has been completely dissolved.

iv. Autoclave solution and equilibrate to 50°C.

5. Aseptically add the agarose solution to the BSKII+antibiotics+rabbit serum. Cap and

mix well by swirling the solution. All of the following steps where cells and/or

medium is manipulated must be performed aseptically under a biological safety hood.

6. Pipette 15mL of the molten medium into three sterile 100x15mm Petri dishes

(Fisher# FB0875712) for each transformation performed. Re-cap bottle of

BSKII/agarose and equilibrate to 42°C in a water bath.

7. While the plates are solidifying and BSKII/agarose is equilibrating, pellet

transformed cells at 3,000 x g for 30min at room temperature. Cells can be examined

beforehand under a microscope to examine health after outgrowth.

8. Remove supernatant from transformed cells using a serological pipette except for

approximately 1mL of liquid. Resuspend the cells completely in the 1mL of

remaining supernatant. Ensure that the plates have solidified and that the

BSKII/agarose is at 42°C before proceeding.

9. Label each plate on the outside rim with the date, the cell background, the DNA used

for transformation, and one of the following percentages: 1%, 10%, 89%.

10. Label three fresh 15mL Falcon tubes with 1%, 10%, and 89%. To each, add 10µL,

100µL, or 890µL of the resuspended transformed cells (respectively).

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11. Add 12mL of BSKII/agarose to one tube of cells, mix by pipetting, and layer

cells/BSKII/agarose over the pre-poured plate that corresponds to the cell

background, transformation and percentage. Avoid bubbles, if possible.

12. Repeat Step 11 for each tube and each transformation, using a fresh serological

pipette for each plate. Allow plates to solidify completely under the hood (≤30min).

13. Incubate plates lid-side-up at 34°C in a humidified 5% CO2 atmosphere. The time

until colonies appear depends on the genetic background of the cells and the nature of

the DNA introduced, with 1-3 weeks being commonplace.

5.6 Surface Proteolysis of B. burgdorferi Using Proteinase K (180)

1. Inoculate 45mL of complete BSK-II (with antibiotics, if necessary) in a 50mL sterile

tube at 1:500 with the desired strain and incubate at 34°C until the culture reaches

late-logarithmic growth phase.

2. Transfer 6mL of the culture to a fresh 15mL tube and harvest at 3,000 x g for 25min,

room temperature. Remove the supernatant using a pipette.

3. Wash the cells once by resuspending gently in 6mL of sterile, room temperature

phosphate buffered saline (pH 7.4) + 5mM MgCl2 (PBS/Mg).

4. Re-pellet the cells at 3,000 x g for 20min. Remove the supernatant using a pipette.

5. Resuspend the cell pellet gently but completely in 300µL of PBS/Mg. Divide the cell

suspension into two 144µL aliquots using fresh 1.5mL microcentrifuge tubes. To

one, add 6µL of Milli-Q dH2O as a control. To the other, add 6µL of 5mg/mL

Proteinase K (Invitrogen# AM2544) in Milli-Q dH2O for 200µg/mL final

concentration. Mix gently by flicking the sides of the tubes.

96

6. Incubate the cells at room temperature for one hour.

7. Add 8µL of 100mM PMSF in DMSO (Sigma-Aldrich# 78830-5G, D2650-5X10ML)

to each sample for approximately 5mM final concentration. Mix thoroughly by

flicking tubes.

8. Pellet cells at 8,000 x g for 10 minutes, room temperature. Completely remove

supernatant using a 200µL pipette.

9. Resuspend each cell pellet in approximately 20µL final volume of Milli-Q dH2O.

Add 5µL of 1M DTT (Fisher# BP172-5, freshly made in Milli-Q dH2O) and 25µL of

2X SDS-PAGE Sample Buffer (Formulated as per Sigma-Aldrich# S3401 but without

β-mercaptoethanol). Mix by flicking tube and cap tightly.

10. Boil samples for 5 minutes and store at -20°C until needed.

5.7 Surface Proteolysis of B. burgdorferi Using Proteinase K (187, 235)

1. Inoculate 45mL of complete BSK-II (with antibiotics, if necessary) in a 50mL sterile

tube at 1:500 with the desired strain and incubate at 34°C until the culture reaches

late-logarithmic growth phase.

2. Transfer 6mL of the culture to a fresh 15mL tube and harvest at 3,000 x g for 25min,

room temperature. Remove the supernatant using a pipette.

3. Wash the cells once by resuspending gently in 6mL of sterile, room temperature

phosphate buffered saline (pH 7.4) + 5mM MgCl2 (PBS/Mg).

4. Re-pellet the cells at 3,000 x g for 20min. Remove the supernatant using a pipette.

5. Resuspend the cell pellet gently but completely in 300µL of PBS/Mg. Divide the cell

suspension into two 142.5µL aliquots using fresh 1.5mL microcentrifuge tubes. To

97

one, add 7.5µL of Milli-Q dH2O as a control. To the other, add 7.5µL of 20mg/mL

Pronase (Roche# 10165921001) in Milli-Q dH2O for 1mg/mL final concentration.

Mix gently by flicking the sides of the tubes.

6. Incubate the cells at 37°C for two hours.

7. Add the following to each sample. Mix by flicking the tubes gently.

i. 48.8µL of PBS/Mg

ii. 0.4µL of 0.5M EDTA (Invitrogen# 15575020)

iii. 0.4µL of 100mM PMSF in DMSO (Sigma-Aldrich# 78830, D2650)

iv. 0.4µL of 400mM Pefabloc SC (Sigma-Aldrich# 76307)

8. Pellet cells at 8,000 x g for 10 minutes, room temperature. Completely remove

supernatant using a 200µL pipette.

9. Resuspend each cell pellet in approximately 20µL final volume of Milli-Q dH2O.

Add 5µL of 1M DTT (Fisher# BP172-5, freshly made in Milli-Q dH2O) and 25µL of

2X SDS-PAGE Sample Buffer (Formulated as per Sigma-Aldrich# S3401 but without

β-mercaptoethanol). Mix by flicking tube and cap tightly.

10. Boil samples for 5 minutes and store at -20°C until needed.

5.8 Membrane Fractionation of B. burgdorferi (30, 181)

1. Inoculate two 250mL cultures of complete BSK-II (with antibiotics, if necessary) using

250µL of a late-logarithmic growth phase culture of each desired strain.

2. Incubate at 34°C until the culture reaches early exponential phase, about 5x106 cells/mL.

At this point, the culture medium should still have its original color and there should not

be visible clumps of cells at the bottom of the bottle. Do not let the culture overgrow.

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3. Prepare solutions for experiment:

a. Prepare 1L of 25mM citrate buffer by dissolving 4.8g of anhydrous citric acid into

800mL of Milli-Q dH2O, adjusting the pH to 3.2 using freshly made 1M NaOH,

and adjusting the volume to 1L using Milli-Q dH2O. Pre-rinse all glassware to be

used thoroughly with Milli-Q dH2O. Divide the preparation of citrate buffer into

two 500mL aliquots.

b. Prepare 500mL of citrate buffer + 0.1% BSA by adding 0.5g of Probumin

BSA(EMD Millipore# 810037) and mixing until dissolved.

c. Prepare the following sucrose solutions in 50mL conical tubes. Each solution will

eventually fully dissolve at room temperature with periodic mixing. Chill on ice

when dissolved.

i. 32g of 25% (w/w) sucrose in citrate buffer

ii. 40g of 42% (w/w) sucrose in citrate buffer

iii. 14g of 56% (w/w) sucrose in citrate buffer

4. Transfer each culture into pre-rinsed 500mL centrifuge bottles and harvest at 3,000 x g

for 25min at room temperature. Decant supernatant.

5. Wash each pellet by resuspension in 250mL of phosphate buffered saline (PBS) + 0.1%

(w/v) Probumin BSA.

6. Re-pellet cells at 3,000 x g for 20min at room temperature. Decant supernatant.

7. Resuspend each pellet completely (no visible particulates) in 45mL of ice-cold 25mM

citrate buffer + 0.1% BSA and transfer to fresh 50mL conical tubes. Seal with Parafilm.

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8. Incubate at room temperature for two hours on a rocker. Every 30 minutes, vortex each

tube for 30 seconds. During this time, pre-chill the XPN-80 ultracentrifuge and SW32Ti

rotor to 4°C.

9. Divide each cell suspension evenly between two pre-rinsed SS-34 tubes and pellet at

20,000 x g for 30min, room temperature.

10. While the cell suspensions are pelleting, pour the discontinuous sucrose gradients in

Beckman UltraClear tubes (#344058). Sucrose solutions must be ice-cold. UltraClear

tubes are not wettable; therefore, the lightest (25%) layer must be poured first and can be

added using a serological pipette. The 42% and 56% layers must then be added in that

order by pipetting underneath the previous layer, displacing the lighter layer upwards.

Use disposable syringes (BD# 302832, 309604) and a blunt-tip syringe needle (Sigma-

Aldrich# Z261351-1EA) to underlay each solution, ensuring crisp interfaces between

each layer. Label the outside of each gradient with a Sharpie and store at 4°C until the

previous step’s centrifugation is finished.

i. 12.5mL of 25% (w/w) sucrose

ii. 15.5mL of 42% (w/w) sucrose

iii. 5mL of 56% (w/w) sucrose

11. Decant the tubes and place on ice. Pool cell pellets from matching samples by

resuspending completely (no visible particulates) in 5mL of ice-cold citrate buffer +

0.1% BSA.

12. Using a 10mL syringe and an 18½G needle, slowly and carefully overlay each cell

suspension over the labeled gradients. Insert each gradient into opposing SW32Ti

swinging buckets (e.g. buckets #’s 1 and 4) using tweezers to slowly drop the gradient

100

into place. Add the caps to the buckets and balance the opposing rotors to within 0.01g,

using ice-cold citrate buffer + 0.1% BSA to make up any difference.

13. Place all six buckets into the pre-chilled SW32Ti rotor in their designated places. Run

the ultracentrifuge at 100,000 x g for 20 hours, 4°C.

14. Immediately remove the gradients from the ultracentrifuge without disturbing the

samples. Leave the ultracentrifuge on and cooled for the following steps.

15. Visualize each gradient. There should be a faint “haze” about 2/3rd of the way up from

the bottom, at the interface between the 25% and 42% solutions. This is the outer

membrane vesicle (OMV) fraction. Towards the bottom of the tube there should be an

obvious, flat disc at the interface between the 42% and 56% solutions. This is the

protoplasmic cylinder (PC) fraction.

16. Using a fresh 10mL syringe + 18½G needle for each layer, needle-aspirate the OMV and

PC fractions from each sample into fresh 50mL conical tubes on ice.

17. Dilute each fraction to approximately 36mL using ice-cold PBS (pH 7.4), which should

result in at least a five-fold dilution of the aspirated sample. Vortex each sample

thoroughly.

18. Prepare the two fractions as follows:

a. Pellet the OMV fractions by transferring the diluted samples to fresh UltraClear

tubes and balancing using ice-cold PBS (pH 7.4). Run in the ultra-centrifuge at

150,000 x g for 4 hours, 4°C. Immediately remove the sample at the end of the

run and carefully decant. The pelleted OMV samples should be visible as

whitish, faint, glassy pellets at the bottom of the tubes. Resuspend each OMV

pellet in 100µL of PBS (pH 7.4) + 1mM PMSF and transfer to fresh 1.5mL

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microcentrifuge tubes. Use a portion of each to prepare SDS-PAGE samples as

was done in Section 5.6. Store the boiled SDS-PAGE samples at -20°C and the

unprocessed OMV suspension at -80°C.

b. Pellet the PC fraction by transferring each to a fresh SS-34 tube and centrifuging

at 10,000 x g for 20min, 4°C. Decant supernatant. Resuspend each cell

completely in 1mL of PBS (pH 7.4) + 1mM PMSF and transfer to fresh 1.5mL

microcentrifuge tubes. Use a portion of each to prepare SDS-PAGE samples as

was done in Section 5.6. Store the boiled SDS-PAGE samples at -20°C and the

unprocessed PC suspension at -80°C.

19. Use equal volumes of each sample for SDS-PAGE analysis.

5.9 Triton X-114 Extraction of Hydrophobic B. burgdorferi Proteins (186)

1. Inoculate two 45mL volumes of complete BSK-II (with antibiotics, if necessary) in

sterile 50mL conical tubes using 90µL each of the desired strain. Incubate at 34°C until

cultures have reached late-logarithmic growth phase. Alternatively, protease-treated cells

can be extracted following Step 8 of Sections 5.6 and 5.7 as long as the volumes are

scaled-up appropriately (in this case, skip to Step 5).

2. Harvest cultures at 3,000 x g for 30min at room temperature. Remove supernatant by

pipetting.

3. Wash each cell pellet by resuspension in 45mL of PBS (pH 7.4) + 5mM MgCl2

(PBS/Mg). Re-pellet at 3,000 x g for 20min, room temperature. Remove supernatant by

pipetting.

4. Repeat Step 3 once more.

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5. Pool cell pellets by resuspension in 1mL of ice-cold 2% (v/v) Triton X-114 in PBS (pH

7.4) + 0.002% (w/v) bromophenol blue. Transfer cell suspension to a fresh 1.5mL

microcentrifuge tube.

6. Rotate cells overnight on a rotisserie at 4°C.

7. Centrifuge sample at 12,000 x g for 30min, 4°C to remove Triton X-114 insoluble

material. Transfer supernatant to a fresh microcentrifuge tube. The insoluble pellet can

be saved for later SDS-PAGE analysis. Optionally, supernatant can be spun again at

12,000 x g for 15min, 4°C to remove remaining traces of insoluble material. In this case,

transfer supernatant again to a fresh tube.

8. Incubate sample at 37°C for 15min to phase-partition the Triton X-114 solution.

9. Centrifuge the sample at 16,000 x g for 15min at room temperature. The resulting

sample should have an upper, faintly blue aqueous phase and a lower, dark blue detergent

phase. Carefully transfer the upper aqueous phase (Sample AQ) to a fresh

microcentrifuge tube without disturbing the lower detergent phase (Sample DE).

10. Wash the samples three times as follows. The washing process should progressively

make Sample AQ less blue and Sample DE more blue.

a. Add 100µL of ice-cold 10% (v/v) Triton X-114 in PBS (pH 7.4) + 0.002% (w/v)

bromophenol blue to Sample AQ and vortex. Add 500µL of ice-cold 1% (v/v)

Triton X-114 in PBS (pH 7.4) + 0.002% (w/v) bromophenol blue to Sample DE

and vortex.

b. Incubate for 15min at 37°C to phase partition the samples.

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c. Centrifuge at 16,000 x g for 15min at room temperature. Transfer the aqueous

phase of Sample AQ to a fresh tube and discard the pelleted detergent. Remove

the aqueous supernatant from Sample DE and retain the detergent phase.

11. Add ice-cold 1% (v/v) Triton X-114 in PBS (pH 7.4) + 0.002% (w/v) bromophenol blue

to Sample DE to a final volume of 500µL and vortex.

12. Divide Sample AQ and Sample DE evenly between four 1.5mL microcentrifuge tubes

each. Add four volumes of 100% acetone (chilled to -20°C) to each sample and vortex.

13. Incubate samples overnight at -20°C.

14. Centrifuge samples at 16,000 x g for 30min, 4°C. Discard supernatant.

15. Wash samples by adding 1mL of 80% (v/v) acetone in Milli-Q dH2O (chilled to -20°C) to

each sample and inverting gently several times.

16. Centrifuge at 16,000 x g for 15min, 4°C. Carefully remove the supernatant by pipetting.

Spin the samples briefly in a centrifuge to collect residual acetone at the bottom of the

tubes, then remove carefully with a 20µL pipette without disturbing the protein pellets.

17. Allow the samples to air-dry on the bench with the caps open for 10min.

18. Pool like samples by resuspension in a suitable buffer and use a fraction for SDS-PAGE

sample preparation (see Section 5.6). If the samples do not easily dissolve into solution,

allow them to incubate with buffer in sealed tubes at 4°C for at least one hour before

resuspension. Store unprocessed samples long-term at -80°C and SDS-PAGE samples at

-20°C.

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Chapter 6: In-depth Analysis of Selected B. burgdorferi Lipoproteins

Lipoproteins are essential to the survival and pathogenesis of B. burgdorferi; however,

many of these have not been functionally characterized through experimentation. In order to

better understand the biological role of the B. burgdorferi lipoproteome, lipoproteins were

analyzed in silico using a variety of methods. Functional prediction using in silico analysis

generates testable hypotheses regarding the B. burgdorferi lipoproteome and can guide future

investigation of under-studied proteins. Of note is that lipoproteins were analyzed only from the

essential genetic elements, the linear chromosome and the circular plasmid cp26, as these

elements are the only ones common to all isolates of B. burgdorferi and, therefore, most likely to

contain essential biosynthetic factors (236). Initially, the full primary sequence of each

lipoprotein was analyzed with the HMMER-based HMMSCAN using all available databases and

the program’s default search settings (237). The top-scoring (by E-value) protein domain/family

for each lipoprotein is reported in Table 4, with relevant domain/family accession numbers and

descriptions given. Several lipoproteins were predicted to contain tetratricopeptide repeat (TPR)

domains, and the identity of these lipoproteins as putative TPR proteins was confirmed by

analysis using TPRpred and is recorded in Table 5 (238). Lipoproteins with no statistically

significant hits reported by HMMSCAN were then analyzed further using their predicted mature

sequence with HHPred, which detects remote homology based on HMM profile-profile

comparison between a multiple sequence alignment of query and deposited PDB structures

(239). HHpred is able detect proteins that share tertiary structural elements despite significant

differences in primary sequences. Table 6 reports the top-scoring PDB hit for each analyzed

lipoprotein. Several lipoproteins from this table were then selected for in-depth analysis, and a

hypothetical in vivo function was formulated based on all available data.

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Table 4. Analysis of Lipoprotein Genes on Essential Genetic Elements by HMMSCAN. Lipoprotein-encoding genes from the essential genetic elements of B. burgdorferi, the chromosome and the circular plasmid cp26, were analyzed using HMMSCAN (236, 237). Genes are reported with the top profile hit by lowest E-value. “SF#” = Superfamily number (superfam.org). Interval corresponds to amino acid region of the unprocessed protein. Genes without a significant hit are reported with “None, n/a.”

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Top Hit ID Description Interval E-value BB0028 None n/a n/a n/a BB0141 TIGR01730 Efflux transporter, RND family, MFP subunit 114-306 1.8e-29 BB0144 PF04069.11 Substrate binding domain of ABC-type glycine

betaine transport system 26-272 8.7e-60

BB0155 None n/a n/a n/a BB0158 None n/a n/a n/a BB0171 SF# 48452 TPR-like 28-144 3.2e-20 BB0193 None n/a n/a n/a BB0213 None n/a n/a n/a BB0215 SF# 53850 Periplasmic binding protein-like II 29-278 1.7e-56 BB0224 None n/a n/a n/a BB0227 None n/a n/a n/a BB0298 SF# 48452 TPR-like 46-211 1.1e-14 BB0323 PF01476.19 LysM domain 327-375 2.3e-08 BB0324 SF# 48452 TPR-like 25-115 2.6e-07 BB0328 PIRSF002741 Peptide binding protein, MppA type 23-528 1.2e-102 BB0329 PIRSF002741 Peptide binding protein, MppA type 14-520 1.6e-102 BB0330 PIRSF002741 Peptide binding protein, MppA type 6-533 3.4e-101 BB0352 None n/a n/a n/a BB0365 None n/a n/a n/a BB0382 PF02608.13 ABC transporter substrate-binding protein PnrA-like 27-328 9.0e-107 BB0384 PF02608.13 ABC transporter substrate-binding protein PnrA-like 29-343 3.1e-107 BB0385 PF02608.13 ABC transporter substrate-binding protein PnrA-like 46-348 6.1e-107 BB0398 SF# 48452 TPR-like 53-120,

158-190 5.9e-09

BB0456 None n/a n/a n/a BB0460 None n/a n/a n/a BB0475 None n/a n/a n/a BB0536 PF00675.19 Insulinase (Peptidase family M16) 45-183 6.3e-27 BB0542 SF# 48452 TPR-like 35-174 7.6e-16 BB0628 None n/a n/a n/a BB0652 TIGR01129 Protein-export membrane protein SecD 138-562 5.5e-141 BB0664 None n/a n/a n/a BB0689 PF00188.25 Cysteine-rich secretory protein family 34-148 3.1e-12 BB0758 None n/a n/a n/a BB0806 SF# 50998 Quinoprotein alcohol dehydrogenase-like 225-499 1.1e-10 BB0823 None n/a n/a n/a BB0832 None n/a n/a n/a BB0844 None n/a n/a n/a BBB08 None n/a n/a n/a BBB09 None n/a n/a n/a BBB25 None n/a n/a n/a BBB27 None n/a n/a n/a

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Table 5. Analysis of putative B. burgdorferi Tetratricopeptide Repeat (TPR) Lipoproteins. Lipoproteins identified in Table 4 were further analyzed by TPRpred to determine the probability of being a true TPR protein (238).

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TPR Probability Per-protein P-value BB0171 100.00% 3.2e-22 BB0298 100.00% 1.7e-16 BB0324 84.53% 5.7e-09 BB0398 99.87% 1.7e-12 BB0542 100.00% 8.9e-17

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Table 6. Remote Homology Analysis of B. burgdorferi Lipoproteins. Lipoproteins without a detectable family/domain or with a hit resulting in an unclear function after HMMSCAN analysis (see Table 4) were analyzed by HHPred (239). Searches were performed against PDB profiles for conserved protein folds using the predicted mature sequence of the lipoprotein. The top-scoring hit by Probab. is reported for each lipoprotein, along with a description of the hit and the hit’s score. Lipoproteins without a significant hit are denoted by “n/a.”

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Top PDB# Description Probab. BB0028 n/a n/a n/a BB0155 3WW9_A Pizza6 self-assembling, synthetic β-propeller protein 99.81 BB0158 n/a n/a n/a BB0193 n/a n/a n/a BB0213 n/a n/a n/a BB0224 4HVZ_A SIMPL domain protein, BP26, Brucella abortus 99.42 BB0227 3CYG_A DUF3298/4163 protein, Fnod_1146, Fervidobacterium nodosum Rt17-B1 100.00 BB0352 2ZYO_A Cyclo/maltodextrin solute-binding protein, Thermoactinomyces vulgaris 100.00 BB0365 n/a n/a n/a BB0456 n/a n/a n/a BB0460 3CYG_A DUF3298/4163 protein, Fnod_1146, Fervidobacterium nodosum Rt17-B1 100.00 BB0475 n/a n/a n/a BB0628 2NC8_A LppM, Rv2171, Mycobacterium tuberculosis 94.70 BB0664 2PLG_A YbjN-like protein T110839, Synechococcus elongatus 97.25 BB0758 n/a n/a n/a BB0806 4A2L_A Hybrid two-component system protein BT4663, Bacteroides

thetaiotaomicron 99.97

BB0823 2KT8_A SH3 domain protein CPE1231, Clostridium perfringens 96.44 BB0832 3K8G_A Outer membrane lipoprotein TP0453, Treponema pallidum 96.11 BB0844 n/a n/a n/a BBB08 n/a n/a n/a BBB09 n/a n/a n/a BBB25 n/a n/a n/a BBB27 n/a n/a n/a

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The first lipoprotein to be investigated in-depth is the product of the gene bb0155. This protein

is well conserved only in the Borrelia genus based on analysis of its primary sequence using

HMMER, although it appears to be somewhat conserved in certain related spirochetes such as

Treponema (237). It may be transcribed as part of an operon with the downstream genes bb0156

and bb0157, although these two gene products are likewise poorly conserved outside of B.

burgdorferi (31, 32). A review of microarray data showed that bb0155 appears not to be

regulated by the Rrp2/RpoN/RpoS, BosR, or the Hk1/Rrp1 pathways (240). Additionally, it was

found that bb0155 was expressed in fed larvae, fed ticks, rat dialysis membrane chambers

(DMCs; a facsimile for vertebrate infection), and in vitro; however, expression was not found to

be significant different in any of the environments (19). Along with the microarray review

study, this indicates that bb0155 is likely a constitutively expressed gene. A different study that

examined the expression of genes in vitro versus in mice showed that bb0155 was expressed

after growth in culture medium and in severe combined immunodeficiency mice, but not in

immunocompetent C3H mice (241). In addition, transfer of ear tissue from infected to

uninfected mice led to transient expression of bb0155 at the 11-day and 22-day timepoints, but

no expression at the 33-day timepoint. It is possible that, based on these two studies, bb0155 is

down-regulated or selected against during persistent vertebrate infection. Interestingly, passive

immunization of mice resulted in down-regulation of most lipoprotein genes but not bb0155.

Taken together, these gene regulation data indicate that the expression of bb0155 is complex, and

its expression may be an interplay of multiple factors. A genome-wide transposon mutagenesis

study of B. burgdorferi failed to recover any bb0155 insertion mutants (bb0156 and bb0157

mutants were recovered), although a separate transposon mutagenesis study found that

insertional mutagenesis of bb0155 negatively impacted the ability of the bacterium to survive on

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maltose as the primary carbon source (37, 242). This may suggest that BB0155 plays a role in

the metabolism of maltose or in the regulation of maltose-inducible genes in the organism.

Analysis using InterPro reveals that the protein contains a predicted six-bladed, β-propeller

(TolB-like) domain, IPR#011042 (243). β-propellers are a structural domain ubiquitous

throughout the domains of life, with varying numbers of “blades” in the propeller and diverse

functional roles (244, 245). Six-bladed β-propellers have been shown to act as structural

proteins, hydrolases, and ligand-binding proteins (amongst other roles). Further analysis of

BB0155 using the remote homology prediction server HHPred against a PDB profile database

revealed significant homology to NHL domain, six-bladed β-propeller proteins (SCOPe#

d1q7fa_, b.68.9.1; Probab=99.93, E-value=1.9e-21) (239). The best “hits” to eubacterial crystal

structures were the C-terminal domain of the Bacteroides thetaiotaomicron protein BT3679

(PDB# 3HRP_A) and the C-terminal domain of the Bacteroides ovatus protein

BACOVA_00264 (PDB# 4HW6_A). In-depth structural analysis of BB0155 using the I-

TASSER web server found striking homology to the Brain tumor protein of Drosophila

melanogaster (PDB# 1Q7F), a 6-bladed, NHL repeat β-propeller. As shown in Figure 11, the

predicted structure of BB0155 and PDB#1Q7F_B align closely, matching all six blades of their

β-propellers. 1Q7F belongs to the CATH superfamily 2.120.10.30 (“TolB, C-terminal domain”),

which contains members with diverse functional roles. Although the diverse functions of β-

propeller proteins make it impossible to ascertain the biological function of BB0155 without

further experimental data, it is likely that the inner membrane localization of BB0155 means that

it does not regulate translation in the same way that 1Q7F does (27, 246). Future studies will

need to focus on the exact biological role of

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Figure 11. Structural Analysis of BB0155 using I-TASSER. The predicted mature sequence of BB0155 was analyzed using the I-TASSER web server with the default settings (247). PDB# 1Q7F_B, belonging to the NHL domain Brain tumor protein of Drosophila melanogaster, was calculated to be the closest structural analog by both TM-score and RMSD. The structure of 1Q7F_B was then superimposed onto the predicted tertiary structure of BB0832, with the rainbow-colored secondary structure cartoon representing BB0832 and the purple backbone trace representing 1Q7F_B.

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BB0155 in B. burgdorferi and whether its role relating to growth in maltose-containing medium

is a direct or indirect effect of its disruption.

The putative lipoprotein BB0352 was similarly studied in detail. It is located in opposite

orientation with the neighboring genes bb0351 and bb0353, indicating that it likely is not part of

an operon with surrounding genes (31, 32). No conserved domains or families were detected

after a search using InterPro; likewise, a HMMER search found that only the Borrelia genus

contains identifiable homologs at the primary sequence level (237, 243). Transposon

mutagenesis experiments indicate that the gene may be essential: one study did not recover any

transposon mutants, while another only recovered a single mutant 3% from the end of the gene

(37, 242). Disruption of a gene within the last 10% of the reading frame is less likely to prevent

the expression of a functional protein (248); this indicates that the single transposon insertion of

bb0352 may in fact result in a protein that is still biologically active. Gene regulation studies

show that the gene may be part of the Rrp2/RpoN/RpoS regulon, with Rrp2 possibly the only

factor responsible for its expression (240, 249). However, other studies found that bb0352 was

not differentially regulated in fed tick environments or in DMCs (19). As the Rrp2/RpoN/RpoS

transcriptional cascade has often been associated with adaptation to the vertebrate host

environment, and because there seems to be no differential regulation in the tick vs. vertebrate

environments, these data suggest that other, unidentified factors may be additionally involved in

bb0352 gene regulation. Structural analysis of BB0352 reveals that the protein bears significant

homology (E-value < 1e-30) to sugar substrate binding proteins of ABC transporters (e.g. PDB#

2ZYO_A). As shown in Figure 12, analysis of a MODELLER-constructed BB0352 PDB using

the protein-ligand modelling server COACH demonstrates that BB0352 likely binds saccharides

such as glucose, maltose, and trehalose (FINDSITE, C-score=0.70, Cluster size=30, Cluster

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representative=PDB#1A7L) (250, 251). This could suggest that BB0352 similarly acts as a

carbohydrate SBP in B. burgdorferi. However, BB0352 was found to localize to the surface of

the cell’s outer membrane, which means that it would not have access to ATP, and does not exist

in an operon with other ABC cassettes (27, 31, 32). Rather than acting in the acquisition of

nutrients, it could be that BB0352 binds sugars in order to mediate B. burgdorferi adhesion to

host tissues. Many proteins are glycosylated with diverse carbohydrates, including galactose

modification of collagen and xylose modification of proteoglycans, and such a modification

could provide an attachment point for B. burgdorferi (252). Similar strategies have been

reported for E. coli and Salmonella enterica, which bind to the extracellular matrix through

oligomannoside modifications (253). Additional support of this hypothesis is found the short

tether of BB0352. Secondary structure prediction of the BB0352 mature sequence using

PSSPRED shows that it contains a four-residue tether, reminiscent of the similarly short tether

region of decorin binding protein A (DbpA; PDB# 4ONR) (228). As the length of a surface

lipoprotein’s tether has been hypothesized to correlate with its biological role, it is quite possible

that, like DbpA, BB0352 acts as a B. burgdorferi adhesin (25). Although further

experimentation is needed in order to clarify the biological role of BB0352, the significant

homology to known sugar-binding proteins and predicted propensity to bind multiple types of

carbohydrates can help guide future study of this protein’s role in B. burgdorferi.

Finally, the putative lipoprotein BB0832 was selected for characterization. The gene

encoding this protein is located on the chromosome in a potential operon with the neighboring

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Figure 12. COACH Analysis of BB0352 reveals a preference for carbohydrate binding. A tertiary structure of BB0352 was constructed by MODELLER using aligned HHPred-identified homologs (239, 251). The model was built using the automatic selection of multiple templates. The resulting model (as a PDB) was then analyzed using COACH to determine possible ligand binding based on previously characterized protein-ligand structures deposited to the PDB (250). The top-scoring hit by C-score was generated by the FINDSITE module, yielding a 30-member cluster represented by Chain A of PDB# 1A7L and a C-score of 0.70. Cluster members bound a variety of saccharides, including maltose, dextrose, trehalose, and maltotetraose. The complete list of cluster member PDBs and the input BB0352 PDB file are available upon request. The predicted binding pocket for maltose (PDB# 1A7L) and coordinating residues of BB0352 are shown above.

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genes encoding isoleucine-tRNA synthetase (bb0833) and the C subunit of the ATP-dependent

Clp protease (bb0844) (31, 32). No transposon mutants of bb0832 were recovered in two

separate studies, suggesting that the gene is essential for in vitro growth (37, 242). Analysis of

microarray data shows that bb0832 is not regulated by any of the Rrp2/RpoN/RpoS, BosR, or

Hk1/Rrp1 pathways (240). It also appears to be expressed in fed larvae, fed nymphs, and DMCs;

yet, its expression is not significantly different between these environments (19). Analysis of

BB0832 using HMMER reveals that no significant homologs exist outside of the Borrelia genus

by primary sequence, and further analysis using HMMSCAN shows that BB0832 does not

contain any identifiable conserved domains or families (237). However, BB0832 was predicted

by HHPred to be homologous to PDB#3K8G_A, TP0453 from Treponema pallidum

(Probab.=96.11, E-value=0.055) (239). TP0453 is the only known lipoprotein of the T. pallidum

outer membrane and is not surface-exposed (254, 255). Interestingly, TP0453 contains

amphipathic α-helices that permit spontaneous membrane integration of nonlipidated TP0453.

Membrane insertion of nonlipidated TP0453 into membranes results in destabilization of the

bilayer and increased membrane permeability. In this way, TP0453 was found to act as a pore-

forming molecule in T. pallidum. TP0453 also contains an internal hydrophobic cavity, with a

large volume exceeding 3500 Å3. The authors of the studies that characterized TP0453

hypothesized that TP0453 may act as a functional homolog for LolB, in that it can spontaneously

insert itself into membranes through its amphipathic α-helices. The authors postulate that this

ability would explain the presence of lipoproteins in the outer membrane of T. pallidum despite

the absence of an identified LolB homolog in the spirochete’s genome. To confirm the

homology between TP0453 and BB0832, the predicted mature sequence of BB0832 was

analyzed using the I-TASSER web server with the default settings (Figure 13) (247).

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Significant homology was found between these two proteins, as a predicted structure of BB0832

was considered structurally analogous to PDB# 3K8G (TP0453) as evidenced by a TM-score of

0.829 and a RMSD of 1.38. Although I-TASSER failed to find any high-confidence ligand-

binding or gene ontology assignments for BB0832, the distinct structural homology between

BB0832 and TP0453 yields the hypothesis that these proteins are analogous and may function

similarly in vivo. The propensity of TP0453 to spontaneously insert into membranes using

amphipathic helices, as well as its large hydrophobic binding pocket, suggest that a B.

burgdorferi homolog with these same properties could play a role in lipoprotein biosynthesis.

Further, the lack of any recovered transposon-disrupted bb0832 mutants suggests that the gene

may be essential for B. burgdorferi growth. Although BB0832 was localized to the inner

membrane using previous localization assays, and therefore might not act as a LolB functional

homolog, it can safely be theorized that it may play an important biosynthetic role in B.

burgdorferi and, as such, warrants further investigation in the future.

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Figure 13. Analysis of BB0832 using I-TASSER. The predicted mature sequence of BB0832

was analyzed using the I-TASSER web server for structural prediction and homology detection

using known PDB structures (247). Significant homology was detected with the T. pallidum

lipoprotein TP0453, PDB# 3K8G. An overlay of the predicted structure of BB0832 (rainbow

cartoon) and PDB# 3K8G (purple backbone trace) is shown above.

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Appendix: Supplemental Figures and Tables

Figure S1. Statistical analyses of quantitative proteomics features for differently-located B. burgdorferi proteins. A. For all proteins detected by MudPIT analysis (Table S1), the difference in sequence coverage (% of protein sequence covered by detected peptides) observed between the proteinase K-treated and control samples were calculated (DiffSeqCov). The ratio in average dNSAF values measured in the control and proteinase K-treated samples were also calculated (dNSAF ratio). The proteins were grouped based on their known or predicted location (7 categories as defined in Table 2 and Table S1) and box-plots of the distribution in DiffSeqCov and dNSAF ratios were built. B. The Mann−Whitney non-parametric U test was performed using OriginPro 9.1 to determine whether or not there were statistically significant differences between the distributions of the DiffSeqCov and dNSAF ratios parameters. The distributions of these parameters for proteins shown to be at the surface of B. burgdorferi B31-A3 cells ("S" proteins in Table 2) were systematically tested against the ranges of DiffSeqCov and dNSAF ratios for proteins shown (Table 2) or predicted to be localized elsewhere.

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x yNull

HypothesisAlternative Hypothesis U Z

Exact Prob>U

Asymp. Prob>U

Table1_S Table1_P-OM F(x) >= G(y) F(x) < G(y) 36 -2.12302 0.01495 0.01688Table1_S Table1_P-IM F(x) >= G(y) F(x) < G(y) 137 -5.90927 3.88E-11 1.72E-09Table1_S SpII F(x) = G(y) F(x) <> G(y) 177 -1.50985 0.13261 0.13108Table1_S SpI F(x) >= G(y) F(x) < G(y) 227 -7.85903 4.82E-19 1.94E-15Table1_S TM F(x) >= G(y) F(x) < G(y) 229 -9.13218 7.40E-27 3.36E-20Table1_S CYT F(x) >= G(y) F(x) < G(y) 1044.5 -10.2737 5.09E-35 4.63E-25SpII Table1_P-OM F(x) = G(y) F(x) <> G(y) 18 -0.21213 0.83916 0.832SpII Table1_P-IM F(x) >= G(y) F(x) < G(y) 64 -2.50294 0.00523 0.00616SpII SpI F(x) >= G(y) F(x) < G(y) 106 -3.34516 2.06E-04 4.11E-04SpII TM F(x) >= G(y) F(x) < G(y) 122 -3.92613 8.60E-06 4.32E-05SpII CYT F(x) >= G(y) F(x) < G(y) 550 -3.76887 2.33E-05 8.20E-05SpI Table1_P-OM F(x) <= G(y) F(x) > G(y) 213 2.28908 0.00872 0.01104SpI Table1_P-IM F(x) = G(y) F(x) <> G(y) 1072 1.62958 0.10298 0.10319SpI TM F(x) = G(y) F(x) <> G(y) 2769 -1.29694 0.19494 0.19465SpI CYT F(x) = G(y) F(x) <> G(y) 10690.5 -0.88955 0.37442 0.37371TM Table1_P-OM F(x) <= G(y) F(x) > G(y) 359 2.68 0.00173 0.00368TM Table1_P-IM F(x) <= G(y) F(x) > G(y) 1894 2.84539 0.00202 0.00222TM CYT F(x) = G(y) F(x) <> G(y) 19291 0.87404 0.38255 0.3821CYT Table1_P-OM F(x) <= G(y) F(x) > G(y) 1288 2.62773 0.00226 0.0043CYT Table1_P-IM F(x) <= G(y) F(x) > G(y) 6603 2.57666 0.00473 0.00499Table1_S Table1_P-OM F(x) <= G(y) F(x) > G(y) 175 2.35912 0.00712 0.00916Table1_S Table1_P-IM F(x) <= G(y) F(x) > G(y) 1270 5.7011 2.78E-10 5.95E-09Table1_S SpII F(x) = G(y) F(x) <> G(y) 300.5 0.88201 0.38061 0.37777Table1_S SpI F(x) <= G(y) F(x) > G(y) 2969 7.77082 0 3.89E-15Table1_S TM F(x) <= G(y) F(x) > G(y) 4763 8.70839 0 0Table1_S CYT F(x) <= G(y) F(x) > G(y) 17343 10.01037 0 0SpII Table1_P-OM F(x) = G(y) F(x) <> G(y) 29 1.20874 0.21578 0.22676SpII Table1_P-IM F(x) <= G(y) F(x) > G(y) 204 2.10662 0.01664 0.01758SpII SpI F(x) <= G(y) F(x) > G(y) 485.5 2.73533 2.50E-03 3.12E-03SpII TM F(x) <= G(y) F(x) > G(y) 766.5 2.76717 2.22E-03 2.83E-03SpII CYT F(x) <= G(y) F(x) > G(y) 2834 2.99067 9.97E-04 1.39E-03SpI Table1_P-OM F(x) >= G(y) F(x) < G(y) 45 -2.13495 0.01464 0.01638SpI Table1_P-IM F(x) >= G(y) F(x) < G(y) 526 -3.06138 9.60E-04 0.0011SpI TM F(x) = G(y) F(x) <> G(y) 3433 0.96365 0.33578 0.33522SpI CYT F(x) = G(y) F(x) <> G(y) 11447.5 -0.05475 0.95654 0.95634TM Table1_P-OM F(x) >= G(y) F(x) < G(y) 62 -2.32492 0.00781 0.01004TM Table1_P-IM F(x) >= G(y) F(x) < G(y) 755 -3.71601 7.23E-05 1.01E-04TM CYT F(x) = G(y) F(x) <> G(y) 16786 -1.23175 0.21829 0.21804CYT Table1_P-OM F(x) >= G(y) F(x) < G(y) 302 -2.02041 2.04E-02 2.17E-02CYT Table1_P-IM F(x) >= G(y) F(x) < G(y) 3177 -3.34438 3.46E-04 4.12E-04

dNSA

F R

atio

(Con

trol

/PK

)D

iffer

ence

in S

eque

nce

Cov

erag

e (P

K -

Con

trol

)

-80

-60

-40

-20

0

20

40

Diff

eren

ce in

Seq

uenc

e C

over

age

(PK

-Cnt

rl)

Tb1_S Tb1_POM Tb1-PIM SpII SpI TM CYT

0

2

4

6

8

10

100200

dNSA

F R

atio

(Cnt

rl/P

K)

Supporting Figure S1: Statistical analyses of quantitative proteomics features for differently-located B. burgdorferi proteinsA. For all proteins detected by MudPIT analysis (Table S2), the difference in sequence coverage (% of protein sequence covered by detected peptides) observed between the proteinase K-treated and control samples were calculated (DiffSeqCov). The ratio in average dNSAF values measured in the control and proteinase K-treated samples were also calculated (dNSAF ratio). The proteins were grouped based on their known or predicted location (7 categories as defined in Table 1 and Table S2) and box-plots of the distribution in DiffSeqCov and dNSAF ratios were built.B. The Mann−Whitney non-parametric U test was performed using OriginPro 9.1 to determine whether or not there were statistically significant differences between the distributions of the DiffSeqCov and dNSAF ratios parameters. The distributions of these parameters for proteins shown to be at the surface of B. burgdorferi B31-A3 cells ("S" proteins in Table 1) were systematically tested against the ranges of DiffSeqCov and dNSAFratios for proteins shown (Table 1) or predicted to be localized elsewhere.

A B

125

Figure S2. Graphical representation of putative lipoprotein genes by localization phenotype and genomic element. Putative lipoproteins were designated as belonging to either an essential genetic element or to a nonessential plasmid based on their encoding genetic element (31, 37, 217-219). Lipoproteins were then assigned a localization phenotype based on the “consensus” localization as designated in Table 1. Pie piece numbers indicate the number of lipoproteins belonging to each class.

126

10

626

Chromosome + cp26

Surface OM, Sub-surface IM

76

25

Nonessential Plasmids

Surface OM, Sub-surface IM

127

Table S1. Detailed peptide and spectral counts of MudPIT-detected proteins from B. burgdorferi B31-A3 either with or without proteolytic shaving. Two replicate B. burgdorferi B31-A3 spirochetes samples subjected to proteinase-K as well as two untreated control samples were analyzed by MudPIT. The MS/MS datasets were searched against a protein database downloaded from the Spirochete Genome Browser, which contains accession numbers of all putative B. burgdorferi B31 ORFs (http://sgb.leibniz-fli.de/cgi/list.pl?ssi=free&c_sid=yes&sid=24&srt=ltg&.submit=Go). Exactly redundant sequences were removed from this database, leaving 1559 non-redundant Bb proteins. After adding usual contaminants and randomizing each protein entry, the total search space was 3472 amino acid sequences. The MS dataset was searched for Methionine oxidation as a differential modification. Combining all 4 runs, proteins had to be identified with at least 2 tryptic peptides of at least 7 amino acid long and/or 2 spectral counts. Proteins that were subsets of others were removed using the parsimony option in DTASelect on the proteins detected after merging all runs. Proteins that were identified by the same set of peptides (including at least one peptide unique to such protein group to distinguish between isoforms) were grouped together, and one accession number was arbitrarily considered as representative of each protein group. Half-tryptic peptides were allowed for the proteinase-K treated individual runs but did not contribute to establishing the master list of identified proteins ("ALL" columns). In each analysis, the following parameters are specified for each protein: Peptide counts (columns end with _P); Total spectral counts (_S); Spectral counts for peptides shared between protein isoforms (_sS); Peptide counts for peptides uniquely matching the protein (_uDP); Spectral counts for peptides uniquely matching the protein (_uS); Spectral counts distributed according to the spectral count contribution of peptides unique to each isoform (_dS); Sequence coverage as a percentage (_SC); Distributed Normalized Spectral Abundance Factors (_dNSAF), calculated based on distributed spectral counts and protein length. The number of times a protein is detected across replicates is listed in the "Detected # Out of" columns. dNSAF are averaged across replicate runs ("dNSAF AVG" columns). Summary statistics on False Discovery Rates (FDR, %) are calculated at the bottom of the table. A total of 621 proteins (excluding contaminants) were detected, including 83 listed in Table 2. Each of the detected proteins was subjected to prediction algorithms to detect potential signal peptidases I & II cleavage sites (LipoP1.0), signal peptide (SignalP4.1), and transmembrane domains (TMHMM2.0). For all detected proteins, the difference in sequence coverage (% of protein sequence covered by detected peptides, merging peptides from the 2 replicates analyzed for each condition) observed between the proteinase K-treated and control samples were calculated. The ratio in average dNSAF values measured in the control and proteinase K-treated samples were also calculated. The proteins were grouped based on their known or predicted location: 51 were tested to be surface (“S”) proteins, 4 were localized to the outer membrane (“P-OM”), and 28 were localized to the inner membrane (“P-IM”). An additional 10 proteins were predicted to contain a signal peptidase II cleavage site by LipoP1.0 (“SpII”), while another 63 were predicted to contain a signal peptide by LipoP1.0 (“SpI”) and/or SignalP4.1. Another 100 proteins were predicted to contain at least 1 transmembrane domain by TMHMM 2.0, but no signal peptide (“TM”), while the remaining 365 proteins were most likely cytoplasmic (“CYT”). The complete MudPIT mass spectrometry dataset (raw files, peak files, search files, as well as DTASelect result files) can be obtained from the MassIVE database via ftp://MSV000080434@massive.ucsd.edu (password ASD06166) and from the ProteomeXchange repository under accession number PXD005617.

128

Dowde

ll_TableS2

Supp

ortin

g Ta

ble

S2. D

etai

led

pept

ide

and

spec

tral

cou

nts

for p

rote

ins

dete

cted

by

Mud

PIT

anal

yses

of B

. bur

gdor

feri

B31

-A3

spiro

chet

es s

ubje

cted

or n

ot to

pro

teol

ytic

sur

face

sha

ving

In e

ach a

naly

sis

, th

e f

ollo

win

g p

ara

mete

rs a

re s

pecifie

d f

or

each p

rote

in:

Peptide c

ounts

(colu

mns e

nd w

ith _

P);

Tota

l spectr

al counts

(_S

);

Spectr

al counts

for

peptides s

hare

d b

etw

een p

rote

in isofo

rms (

_sS

);

Peptide c

ounts

for

peptides u

niq

uely

matc

hin

g the p

rote

in (

_uD

P);

Spectr

al counts

for

peptides u

niq

uely

matc

hin

g the p

rote

in (

_uS

);

Spectr

al counts

dis

trib

ute

d a

ccord

ing to the s

pectr

al count contr

ibution o

f peptides u

niq

ue to e

ach isofo

rm (

_dS

);

Sequence c

overa

ge a

s a

perc

enta

ge (

_S

C);

Dis

trib

ute

d N

orm

aliz

ed S

pectr

al A

bundance F

acto

rs (

_dN

SA

F),

calc

ula

ted b

ased o

n d

istr

ibute

d s

pectr

al counts

and p

rote

in length

.

The n

um

ber

of

tim

es a

pro

tein

is d

ete

cte

d a

cro

ss r

eplic

ate

s is lis

ted in the "

Dete

cte

d #

Out of"

colu

mns.

dN

SA

F a

re a

vera

ged a

cro

ss r

eplic

ate

runs (

"dN

SA

F A

VG

" colu

mns).

Sum

mary

sta

tistics o

n F

als

e D

iscovery

Rate

s (

FD

R, %

) are

calc

ula

ted a

t th

e b

ottom

of

the table

.

A tota

l of

621 p

rote

ins (

exclu

din

g c

onta

min

ants

) w

ere

dete

cte

d, in

clu

din

g 8

3 lis

ted in T

able

1. E

ach o

f th

e d

ete

cte

d p

rote

ins w

as s

ubje

cte

d to p

redic

tion a

lgorith

ms to d

ete

ct pote

ntial sig

nal peptidases I &

II cle

avage s

ites (

Lip

oP

1.0

), s

ignal peptide (

Sig

nalP

4.1

), a

nd tra

nsm

em

bra

ne d

om

ain

s (

TM

HM

M2.0

),

The p

rote

ins w

ere

gro

uped b

ased o

n their k

now

n o

r pre

dic

ted location:

The c

om

ple

te M

udP

IT m

ass s

pectr

om

etr

y d

ata

set (r

aw

file

s, peak f

iles, searc

h f

iles, as w

ell

as D

TA

Sele

ct re

sult f

iles)

can b

e o

bta

ined f

rom

the M

assIV

E d

ata

base v

ia f

tp://M

SV

000080434@

massiv

e.u

csd.e

du (

passw

ord

AS

D06166)

and f

rom

the P

rote

om

eX

change r

epository

under

accessio

n n

um

ber

PX

D005617.

Kno

wn

(Tab

le 1

) an

d/or

pr

edic

ated

su

bcel

lula

r lo

catio

nLo

cus

Prot

eins

In

Gro

upD

escr

iptio

n

B31A

3_C

ontr

ol_

mer

ged-

SeqC

ov

(%)

B31A

3_Pr

oK_m

erge

d-Se

qCov

(%

)

Diff

SeqC

ov

(Con

trol

- Pr

oK) (

%)

B31A

3_C

ntl-T

i dN

SAF

AVG

B31A

3_C

ntl-T

i D

etec

ted

# O

ut o

f 2

B31A

3_P

K-T

i dN

SAF

AVG

B31A

3_P

K-T

i D

etec

ted

# O

ut o

f 2

B31A

3_C

ntl-T

i:B3

1A3_

PK-T

i

B31A

3_C

ont

rol

_1_P

B31A

3_C

ont

rol

_1_S

B31A

3_C

ont

rol

_1_u

S

B31A

3_C

ont

rol

_1_u

DP

B31A

3_C

ont

rol

_1_s

S

B31A

3_C

ont

rol

_1_d

S

B31A

3_C

ont

rol

_1_S

C

B31A

3_C

ontr

ol_1

_dN

SAF

B31A

3_C

ont

rol

_2_P

B31A

3_C

ont

rol

_2_S

B31A

3_C

ont

rol

_2_u

S

B31A

3_C

ont

rol

_2_u

DP

B31A

3_C

ont

rol

_2_s

S

B31A

3_C

ont

rol

_2_d

S

B31A

3_C

ont

rol

_2_S

C

B31A

3_C

ontr

ol_2

_dN

SAF

B31A

3_Pr

oK_1

_P

B31A

3_Pr

oK_1

_S

B31A

3_Pr

oK_1

_uS

B31A

3_Pr

oK_1

_uD

P

B31A

3_Pr

oK_1

_sS

B31A

3_Pr

oK_1

_dS

B31A

3_Pr

oK_1

_SC

B31A

3_P

roK

_1_d

NSA

F

B31A

3_Pr

oK_2

_P

B31A

3_Pr

oK_2

_S

B31A

3_Pr

oK_2

_uS

B31A

3_Pr

oK_2

_uD

P

B31A

3_Pr

oK_2

_sS

B31A

3_Pr

oK_2

_dS

B31A

3_Pr

oK_2

_SC

B31A

3_P

roK

_2_d

NSA

FAL

L_y2

_PAL

L_y2

_S

ALL_

y2_u

S

ALL_

y2_u

DP

ALL_

y2_s

S

ALL_

y2_d

S

ALL_

y2_S

CAL

L_y2

_dN

SAF

Leng

thM

WpI

SB

B_

01

58

BB

_0

15

8an

tig

en

6

8.4

92

3.5

3-4

4.9

60

.00

24

32

0.0

00

24

21

0.3

44

71

56

56

51

50

65

55

.50

.00

20

21

79

09

01

70

90

65

.60

.00

28

54

88

40

82

3.5

0.0

00

42

22

20

28

.82

6.9

E-0

52

11

65

16

52

10

16

56

8.5

0.0

01

52

38

27

26

26

.7

SB

B_

02

13

BB

_0

21

3p

red

icte

d c

od

ing

reg

ion

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02

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9

.22

11

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2.3

0.0

00

11

8.4

E-0

52

1.2

14

29

23

32

03

9.2

20

.00

01

XX

XX

XX

XX

11

11

01

3.2

35

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-05

23

32

03

11

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.00

01

14

66

40

61

86

E-0

52

17

25

76

06

.6

SB

B_

06

89

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_0

68

9p

red

icte

d c

od

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reg

ion

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06

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5

8.0

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0.0

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98

34

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80

34

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.00

16

24

10

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04

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10

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E-0

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14

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06

31

55

17

97

19

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SB

B_

A0

4B

B_

A0

4an

tig

en

1

6.3

10

-16

.31

5.3

E-0

51

00∞

XX

XX

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22

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02

16

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-05

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XX

XX

XX

XX

XX

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22

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28

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B_

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rote

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01

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69

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53

05

40

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02

3X

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XX

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XX

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X3

77

30

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0.1

9.3

E-0

51

62

18

58

99

SB

B_

A1

4B

B_

A1

4con

serv

ed

hyp

oth

etical p

rote

in

19

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6-1

0.7

50

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01

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0.0

00

21

0.6

26

26

11

11

01

9.9

26

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-05

33

33

03

19

0.0

00

19

12

21

02

8.2

60

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02

XX

XX

XX

XX

36

63

06

19

0.0

00

11

12

11

39

50

9.6

SB

B_

A1

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B_

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ter

su

rface p

rote

in A

(osp

A)

90

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66

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53

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53

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81

30

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48

0.0

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6#

##

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27

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67

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04

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03

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17

17

9

SB

B_

A2

4B

B_

A2

4d

ecorin

bin

din

g p

rote

in A

(d

bp

A)

53

.93

30

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-23

.04

0.0

01

26

20

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02

12

5.9

52

61

61

41

46

01

43

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0.0

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10

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50

10

05

05

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01

97

24

42

04

17

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02

53

44

30

42

5.1

0.0

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17

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72

72

11

07

25

3.9

0.0

00

82

19

12

12

14

8.8

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B_

A5

7B

B_

A5

7p

red

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d c

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reg

ion

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A5

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7.7

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42

2

1.8

83

6.4

61

4.5

80

.00

08

82

0.0

00

72

1.2

58

99

62

02

06

02

02

1.9

0.0

00

77

32

52

53

02

51

1.5

0.0

00

98

28

82

08

11

.50

.00

05

62

12

16

02

13

6.5

0.0

00

89

87

47

48

07

44

0.1

0.0

00

83

19

22

22

19

7.9

P-I

MB

B_

06

28

BB

_0

62

8con

serv

ed

hyp

oth

etical p

rote

in

01

3.2

81

3.2

80

00

.00

02

22

0X

XX

XX

XX

XX

XX

XX

XX

X2

22

20

21

3.3

9.9

E-0

52

10

10

20

10

9.9

60

.00

03

43

12

12

30

12

13

.30

.00

01

12

41

27

49

05

.6

P-I

MB

B_

06

52

BB

_0

65

2p

rote

in-e

xp

ort

mem

bra

ne p

rote

in (

s1

3.6

53

8.2

32

4.5

80

.00

01

22

0.0

00

68

20

.17

76

84

99

40

91

0.2

0.0

00

11

31

01

03

01

01

0.4

0.0

00

13

11

21

21

11

02

12

2.5

0.0

00

43

15

67

67

15

06

73

4.5

0.0

00

93

19

10

51

05

19

01

05

35

.80

.00

03

95

86

65

02

88

.9

P-I

MB

B_

06

64

BB

_0

66

4p

red

icte

d c

od

ing

reg

ion

BB

06

64

3

4.3

65

9.9

12

5.5

50

.00

10

71

0.0

01

54

20

.69

83

16

33

33

60

33

34

.40

.00

10

7X

XX

XX

XX

X1

05

55

51

00

55

59

.90

.00

29

35

53

05

22

0.0

00

18

10

93

93

10

09

35

9.9

0.0

00

89

22

72

65

10

5.1

P-I

MB

B_

08

06

BB

_0

80

6p

red

icte

d c

od

ing

reg

ion

BB

08

06

2

4.7

18

.77

-5.9

30

.00

02

42

0.0

00

22

1.2

17

17

10

31

31

10

03

12

4.7

0.0

00

45

22

22

02

6.5

23

E-0

54

12

12

40

12

14

.20

.00

02

84

77

40

71

2.7

0.0

00

11

11

52

52

11

05

22

7.1

0.0

00

22

50

65

82

84

6.7

P-I

MB

B_

08

32

BB

_0

83

2p

red

icte

d c

od

ing

reg

ion

BB

08

32

8

.03

17

.15

9.1

22

.7E

-05

13

.7E

-05

20

.72

97

31

11

10

18

.03

2.7

E-0

5X

XX

XX

XX

X1

11

10

18

.03

4.4

E-0

51

11

10

19

.12

3E

-05

22

22

02

8.0

31

.6E

-05

27

43

11

19

9.6

P-I

MB

B_

A0

3B

B_

A0

3ou

ter

mem

bra

ne p

rote

in

78

.11

75

.15

-2.9

60

.02

12

52

0.0

30

24

20

.70

27

81

95

10

51

01

90

51

07

2.2

0.0

22

29

25

45

44

54

25

04

54

78

.10

.02

02

13

26

10

61

03

20

61

07

2.8

0.0

43

14

24

35

93

59

24

03

59

69

.20

.01

73

32

91

88

11

88

12

90

18

81

85

.80

.02

40

81

69

19

22

25

.4

P-I

MB

B_

A0

5B

B_

A0

5an

tig

en

1

6.0

74

.08

-11

.99

4.4

E-0

52

2.4

E-0

52

1.8

33

33

24

42

04

7.6

77

.1E

-05

11

11

01

8.3

91

.8E

-05

11

11

01

3.3

62

.9E

-05

11

11

01

3.8

42

E-0

53

55

30

51

6.1

2.6

E-0

54

17

48

84

29

.1

P-I

MB

B_

A3

4B

B_

A3

4olig

op

ep

tid

e A

BC

tra

nsp

ort

er

7.5

61

7.2

9.6

44

.3E

-05

25

.8E

-05

20

.74

13

83

20

31

17

3.0

47

.56

4.2

E-0

53

19

31

16

3.0

27

.56

4.3

E-0

54

15

32

12

3.0

21

0.4

6.8

E-0

55

44

32

41

3.0

41

2.7

4.7

E-0

54

89

11

27

81

1.1

12

.54

.5E

-05

52

96

09

93

5.7

P-I

MB

B_

B1

6B

B_

B1

6olig

op

ep

tid

e A

BC

tra

nsp

ort

er

76

.04

75

.47

-0.5

70

.01

75

82

0.0

28

78

20

.61

07

74

71

18

51

18

14

64

11

83

65

.10

.01

64

95

11

31

51

31

15

04

13

15

72

.60

.01

86

74

71

36

71

36

74

70

13

67

70

.40

.03

08

37

21

73

61

73

57

11

17

36

71

.50

.02

67

36

75

55

55

54

86

67

55

55

76

0.0

22

68

53

06

07

66

6.5

P-I

MB

B_

B2

7B

B_

B2

7p

red

icte

d c

od

ing

reg

ion

BB

B2

7

10

.93

8.7

4-2

.19

8.1

E-0

51

6.5

E-0

51

1.2

46

15

12

21

02

10

.98

.1E

-05

XX

XX

XX

XX

11

11

01

8.7

46

.5E

-05

XX

XX

XX

XX

23

32

03

19

.73

.5E

-05

18

32

15

51

9.5

For

all

dete

cte

d p

rote

ins, th

e d

iffe

rence in s

equence c

overa

ge (

% o

f pro

tein

sequence c

overe

d b

y d

ete

cte

d p

eptides, m

erg

ing p

eptides f

rom

the 2

replic

ate

s a

naly

zed f

or

each c

onditio

n)

observ

ed b

etw

een the p

rote

inase K

-tre

ate

d a

nd c

ontr

ol sam

ple

s w

ere

calc

ula

ted. T

he r

atio in a

vera

ge d

NS

AF

valu

es m

easure

d in the c

ontr

ol and p

rote

inase K

-tre

ate

d s

am

ple

s w

ere

als

o c

alc

ula

ted.

Tw

o r

eplic

ate

B. burg

dorf

eri B

31-A

3 s

pirochete

s s

am

ple

s s

ubje

cte

d to p

rote

inase-K

as w

ell

as tw

o u

ntr

eate

d c

ontr

ol sam

ple

s w

ere

analy

zed b

y M

udP

IT.

The M

S/M

S d

ata

sets

were

searc

hed a

gain

st a p

rote

in d

ata

base d

ow

nlo

aded f

rom

the S

pirochete

Genom

e B

row

ser,

whic

h c

onta

ins a

ccessio

n n

um

bers

of

all p

uta

tive B

. burg

dorf

eri B

31 O

RF

s (

http://s

gb.leib

niz

-fli.d

e/c

gi/list.pl?

ssi=

free&

c_sid

=yes&

sid

=24&

srt

=ltg&

.subm

it=

Go).

Exactly r

edundant sequences w

ere

rem

oved f

rom

this

data

base, le

avin

g 1

559 n

on-r

edundant B

b p

rote

ins. A

fter

addin

g u

sual conta

min

ants

and r

andom

izin

g e

ach p

rote

in e

ntr

y, th

e tota

l searc

h s

pace w

as 3

472 a

min

o a

cid

sequences. T

he M

S d

ata

set w

as s

earc

hed f

or

Meth

ionin

e o

xid

ation a

s a

diffe

rential m

odific

ation.

Com

bin

ing a

ll 4 r

uns, pro

tein

s h

ad to b

e identified w

ith a

t le

ast 2 try

ptic p

eptides o

f at le

ast 7 a

min

o a

cid

long a

nd/o

r 2 s

pectr

al counts

. P

rote

ins that w

ere

subsets

of

oth

ers

were

rem

oved u

sin

g the p

ars

imony o

ption in D

TA

Sele

ct on the p

rote

ins d

ete

cte

d a

fter

merg

ing a

ll ru

ns. P

rote

ins that w

ere

identified b

y the s

am

e s

et of

peptides (

inclu

din

g a

t le

ast one p

eptide u

niq

ue to s

uch p

rote

in g

roup to d

istinguis

h b

etw

een isofo

rms)

were

gro

uped togeth

er,

and o

ne a

ccessio

n n

um

ber

was a

rbitra

rily

consid

ere

d a

s r

epre

senta

tive o

f each p

rote

in g

roup. H

alf-t

ryptic p

eptides w

ere

allow

ed f

or

the p

rote

inase-K

tre

ate

d indiv

idual ru

ns b

ut did

not contr

ibute

to e

sta

blishin

g the m

aste

r list of

identified p

rote

ins (

"ALL"

colu

mns).

51 w

ere

teste

d to b

e s

urf

ace (

“S”)

pro

tein

s, 4 w

ere

localized to the o

ute

r m

em

bra

ne (

“P-O

M”)

, and 2

8 w

ere

localized to the inner

mem

bra

ne (

“P-I

M”)

. A

n a

dditio

nal 10 p

rote

ins w

ere

pre

dic

ted to c

onta

in a

sig

nal peptidase II cle

avage s

ite b

y L

ipoP

1.0

(“S

pII

”), w

hile

anoth

er

63 w

ere

pre

dic

ted to c

onta

in a

sig

nal peptide b

y L

ipoP

1.0

(“S

pI”

) and/o

r S

ignalP

4.1

. A

noth

er

100 p

rote

ins w

ere

pre

dic

ted to c

onta

in a

t le

ast 1 tra

nsm

em

bra

ne

dom

ain

by T

MH

MM

2.0

, but no s

ignal peptide (

“TM

”), w

hile

the r

em

ain

ing 3

65 p

rote

ins w

ere

most lik

ely

cyto

pla

sm

ic (

“CY

T”)

.

1

129

Dowde

ll_TableS2

Kno

wn

(Tab

le 1

) an

d/or

pr

edic

ated

su

bcel

lula

r lo

catio

nLo

cus

Prot

eins

In

Gro

upD

escr

iptio

n

B31A

3_C

ontr

ol_

mer

ged-

SeqC

ov

(%)

B31A

3_Pr

oK_m

erge

d-Se

qCov

(%

)

Diff

SeqC

ov

(Con

trol

- Pr

oK) (

%)

B31A

3_C

ntl-T

i dN

SAF

AVG

B31A

3_C

ntl-T

i D

etec

ted

# O

ut o

f 2

B31A

3_P

K-T

i dN

SAF

AVG

B31A

3_P

K-T

i D

etec

ted

# O

ut o

f 2

B31A

3_C

ntl-T

i:B3

1A3_

PK-T

i

B31A

3_C

ont

rol

_1_P

B31A

3_C

ont

rol

_1_S

B31A

3_C

ont

rol

_1_u

S

B31A

3_C

ont

rol

_1_u

DP

B31A

3_C

ont

rol

_1_s

S

B31A

3_C

ont

rol

_1_d

S

B31A

3_C

ont

rol

_1_S

C

B31A

3_C

ontr

ol_1

_dN

SAF

B31A

3_C

ont

rol

_2_P

B31A

3_C

ont

rol

_2_S

B31A

3_C

ont

rol

_2_u

S

B31A

3_C

ont

rol

_2_u

DP

B31A

3_C

ont

rol

_2_s

S

B31A

3_C

ont

rol

_2_d

S

B31A

3_C

ont

rol

_2_S

C

B31A

3_C

ontr

ol_2

_dN

SAF

B31A

3_Pr

oK_1

_P

B31A

3_Pr

oK_1

_S

B31A

3_Pr

oK_1

_uS

B31A

3_Pr

oK_1

_uD

P

B31A

3_Pr

oK_1

_sS

B31A

3_Pr

oK_1

_dS

B31A

3_Pr

oK_1

_SC

B31A

3_P

roK

_1_d

NSA

F

B31A

3_Pr

oK_2

_P

B31A

3_Pr

oK_2

_S

B31A

3_Pr

oK_2

_uS

B31A

3_Pr

oK_2

_uD

P

B31A

3_Pr

oK_2

_sS

B31A

3_Pr

oK_2

_dS

B31A

3_Pr

oK_2

_SC

B31A

3_P

roK

_2_d

NSA

FAL

L_y2

_PAL

L_y2

_S

ALL_

y2_u

S

ALL_

y2_u

DP

ALL_

y2_s

S

ALL_

y2_d

S

ALL_

y2_S

CAL

L_y2

_dN

SAF

Leng

thM

WpI

PB

B_0

536

BB

_053

6zi

nc p

rote

ase

67.3

165

.17

-2.1

40.

0066

92

0.00

993

20.

6733

559

581

581

590

581

60.7

0.00

4671

1088

1088

710

1088

61.1

0.00

877

5351

251

253

051

252

.70.

0065

697

1525

1521

974

1521

64.6

0.01

3310

036

3436

3410

00

3634

71.7

0.00

843

933

1084

586.

3

SpI

IB

B_0

383

BB

_038

3ba

sic

mem

bran

e pr

otei

n A

(bm

pA)

86.7

379

.94

-6.7

90.

0409

72

0.05

328

20.

7689

134

1434

1432

342

1432

77.9

0.03

1243

2286

2286

430

2286

86.7

0.05

074

4316

9116

9143

016

9175

.50.

0596

359

1950

1950

590

1950

74.9

0.04

694

4972

2072

1349

772

1386

.70.

0460

433

936

968

5.3

SpI

IB

B_0

639

BB

_063

9sp

erm

idin

e/pu

tresc

ine

AB

C tr

ansp

o81

.976

.72

-5.1

80.

0166

12

0.01

806

20.

9197

134

630

630

340

630

70.1

0.01

337

4091

891

840

091

881

.60.

0198

543

644

644

430

644

70.7

0.02

212

5359

759

753

059

772

.70.

014

5827

1427

1458

027

1483

.90.

0168

734

840

681

5.9

SpI

IB

B_0

840

BB

_084

0pr

edic

ted

codi

ng re

gion

BB

0840

32

.16

27.7

-4.4

60.

0003

32

0.00

021

21.

5990

313

3636

130

3632

.20.

0004

94

1212

40

1213

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0001

73

77

30

710

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0001

69

1717

90

1727

.70.

0002

616

7070

160

7036

.60.

0002

853

862

131

8.9

SpI

IB

B_A

25B

B_A

25de

corin

bin

ding

pro

tein

B (d

bpB

) 20

.32

9.63

-10.

690.

0001

62

5.4E

-05

22.

9444

42

66

20

610

.70.

0002

42

22

20

215

.58E

-05

11

11

01

9.63

6.4E

-05

11

11

01

9.63

4.4E

-05

310

103

010

20.3

0.00

012

187

2039

89.

6S

pII

BB

_A60

BB

_A60

surfa

ce li

popr

otei

n P

27

79.4

29.

75-6

9.67

0.00

236

27.

3E-0

52

32.3

425

1462

6214

062

48.4

0.00

165

2611

311

326

011

378

0.00

307

22

22

02

9.75

8.6E

-05

12

21

02

5.42

5.9E

-05

3117

817

831

017

879

.40.

0013

927

732

444

9.1

SpI

IB

B_A

66B

B_A

66an

tigen

0

3.89

3.89

00

0.00

004

10

XX

XX

XX

XX

XX

XX

XX

XX

XX

XX

XX

XX

12

21

02

3.89

4E-0

51

22

10

23.

891.

1E-0

541

145

874

9.2

SpI

IB

B_A

73B

B_A

73an

tigen

6.

080

-6.0

85.

1E-0

51

00∞

XX

XX

XX

XX

12

21

02

6.08

5.1E

-05

XX

XX

XX

XX

XX

XX

XX

XX

12

21

02

6.08

1.5E

-05

296

3464

78.

9S

pII

BB

_J08

BB

_J08

pred

icte

d co

ding

regi

on B

BJ0

8 2.

2910

.46

8.17

00

5.3E

-05

10

18

00

80

2.29

XX

XX

XX

XX

XX

XX

XX

XX

X1

22

10

210

.55.

3E-0

52

102

18

2.03

12.8

1.4E

-05

306

3464

08.

9S

pII

BB

_K47

BB

_K47

pred

icte

d co

ding

regi

on B

BK

47

51.2

20

-51.

220.

0005

20

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1539

48.3

0.02

317

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1253

0.01

242

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88.

6TM

BB

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BB

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cons

erve

d hy

poth

etic

al p

rote

in

20.7

16.

43-1

4.28

0.00

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5E-0

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74

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14.3

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32

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0001

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11

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438.

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XX

XX

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1120

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0001

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016

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TMB

B_J

27B

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27pr

edic

ted

codi

ng re

gion

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97.

3-3

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63

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XX

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12

21

02

3.16

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22

22

02

7.3

4E-0

53

1010

30

1011

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3E-0

541

146

456

5.8

TMB

B_K

13B

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nser

ved

hypo

thet

ical

pro

tein

14

.71

43.7

28.9

99.

5E-0

51

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XX

XX

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20

314

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XX

XX

XX

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3030

90

3043

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0010

310

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100

3344

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0003

238

2722

19.

2TM

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252

cons

erve

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poth

etic

al in

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al m

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61.

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8E-0

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3580

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88

40

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567.

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44

10

42.

353.

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1818

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60.

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44

20

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564.

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3310

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576

786

971

9.6

TMB

B_0

091

BB

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1V

-type

ATP

ase

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22.5

319

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3.6E

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0.07

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31

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2.8

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XX

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022

608

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58.

3TM

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8B

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408

PTS

sys

tem

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35.8

43.

360.

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32

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3765

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186

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318

330

318

32.8

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341

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338

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0029

962

567

130

7.5

TMB

B_0

629

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9P

TS s

yste

m

28.1

235

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0013

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178

310

178

36.4

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626

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58.

9TM

BB

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PTS

sys

tem

7

10.8

43.

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32

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20

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30

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466

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066

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122

449

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70.

0002

444

348

761

8.1

TMB

B_0

071

BB

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1pr

edic

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codi

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gion

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11

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63

06

8.37

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99

50

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5816

49.

3TM

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olig

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tide

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C tr

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23.5

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52

05

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22.9

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196

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512

512

012

525

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734

939

418

9.2

TMB

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145

BB

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5gl

ycin

e be

tain

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025.

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9.9E

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10.

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81

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10

45.

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XX

XX

XX

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10

25.

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XX

XX

XX

16

61

06

5.02

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299

3373

59

TMB

B_0

118

BB

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8zi

nc p

rote

ase

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232

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5.95

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92

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299

029

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9612

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26.5

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018

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035

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0008

943

749

741

9.2

TMB

B_0

170

BB

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0pr

edic

ted

codi

ng re

gion

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0170

20

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30.2

19.

980.

0001

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025

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73

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6.89

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3410

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23.3

0.00

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3535

90

3522

0.00

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1494

9414

094

32.3

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278

475

8.8

TMB

B_0

442

BB

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2in

ner m

embr

ane

prot

ein

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16.1

87.

915.

5E-0

52

0.00

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55

20

54.

786.

8E-0

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33

10

33.

494.

1E-0

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66

40

610

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0001

33

99

30

98.

640.

0001

46

2222

60

2213

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8E-0

554

464

002

8.9

TMB

B_0

157

BB

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7pr

edic

ted

codi

ng re

gion

BB

0157

10

.14

10.1

40

0.00

011

20.

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3648

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22

10

210

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0001

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22

10

210

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1X

XX

XX

XX

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55

10

510

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0003

19

91

09

10.1

0.00

014

138

1559

59.

6TM

BB

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7B

B_0

217

phos

phat

e A

BC

tran

spor

ter

5.18

18.6

13.4

22.

3E-0

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1X

XX

XX

XX

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10

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3E-0

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412

124

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18.6

0.00

034

2424

40

2418

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0001

632

836

113

9.3

TMB

B_0

380

BB

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0M

g2+

trans

port

prot

ein

(mgt

E)

4.19

10.1

35.

944.

9E-0

51

0.00

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21.

225

23

32

03

4.19

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XX

XX

XX

11

11

01

1.76

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23

32

03

8.37

5.4E

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47

74

07

12.6

3.3E

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5074

75.

1TM

BB

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B_0

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pher

omon

e sh

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prot

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31.6

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240.

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110

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4521

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123

128

128

230

128

41.6

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404

4567

89.

3TM

BB

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6B

B_0

766

colic

in V

pro

duct

ion

prot

ein

8.64

8.64

00.

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71

0.00

011

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386

18

81

08

8.64

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XX

XX

XX

XX

XX

XX

XX

XX

12

21

02

8.64

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108.

640.

0001

316

218

716

9.1

TMB

B_0

453

BB

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3co

nser

ved

hypo

thet

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pro

tein

0

25.3

625

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00

0.00

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XX

XX

XX

XX

XX

XX

XX

XX

11

11

01

4.29

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298

029

25.4

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030

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0.00

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280

3230

28.

6TM

BB

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5B

B_0

595

pred

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regi

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011

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11.0

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00.

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XX

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XX

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XX

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33

30

311

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33

30

311

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1E-0

520

824

003

9.8

TMB

B_A

01B

B_A

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ved

hypo

thet

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pro

tein

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450

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54.

6E-0

52

00∞

11

11

01

7.45

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11

01

6.21

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XX

XX

XX

XX

XX

XX

XX

XX

22

22

02

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161

1788

79.

5TM

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pred

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75

19.4

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470.

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170.

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22

10

22.

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130

2926

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6316

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469

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89.

2TM

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134

pred

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regi

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34

4.71

3.93

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51

3.1E

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11

11

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XX

XX

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11

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3.93

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XX

XX

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22

22

02

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59.

3TM

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long

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CoA

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32.8

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17.9

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629

261

261

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261

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0009

630

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38.

8TM

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291

flage

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135

135

270

135

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156

965

134

5.5

TMB

B_0

596

BB

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6m

ethy

l-acc

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axis

pro

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29.5

120

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40

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676.

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XX

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4242

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4228

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140

6428

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0001

971

580

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7.6

TMB

B_0

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BB

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ted

codi

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328

207

207

280

207

540.

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257

435

8.7

TMB

B_E

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B_E

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ved

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00

0.00

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XX

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24

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42

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2928

28.

6TM

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4213

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6762

76.

4TM

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pred

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5.65

5.65

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10

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3448

89

TMB

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166

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4.4

TMB

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339

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5.8

TMB

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TMB

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7.9

TMB

B_G

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ved

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37.

142.

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5.5

TMB

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5.3

TMB

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294

5.2

TMB

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00

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12.9

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5.5

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41.4

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5.4

TMB

B_O

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18.9

528

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TMB

B_R

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3747

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11

10

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ved

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00

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XX

XX

XX

XX

XX

XX

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Sig

nalP

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MS

S62

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933

37B

B_0

844

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++

SpI

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3494

516

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R0.

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180

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233

230.

319

230.

741

20.

467

0.38

9N

0.57

Sig

nalP

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MV

raA

SS

∞S

1117

9306

5475

P35

60

naS

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10.0

931

8.21

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0.25

0.25

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0.48

230.

612

230.

929

190.

789

0.69

5Y

0.57

Sig

nalP

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MS

S8.

9386

526

27P

35

60+

++

naS

pI5.

9947

22.

1095

429

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18.0

418

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1i9

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0.21

227

0.43

527

0.95

225

0.86

90.

639

Y0.

57S

igna

lP-n

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P35

S

S85

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232

37P

35

54na

SpI

5.79

867

1.77

267

29-3

018

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18.0

41

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212

270.

434

270.

952

250.

867

0.63

8Y

0.57

Sig

nalP

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MS

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3238

P35

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+na

SpI

7.64

303

1.76

749

33-3

45.

635.

630

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196

270.

415

270.

925

250.

855

0.62

2Y

0.57

Sig

nalP

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MS

S56

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633

37P

35

54+

naS

pII

18.2

273

9.15

461

19-2

0V

0.57

0.57

0o

0.44

524

0.62

624

0.96

316

0.89

90.

754

Y0.

57S

igna

lP-n

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Osp

DS

S22

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4S

1398

980

2829

++

SpI

I10

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17.

1011

218

-19

K3.

333.

220

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228

240.

363

240.

778

40.

596

0.47

2N

0.57

Sig

nalP

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MS

S14

2.69

640

41C

RA

SP

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92+

+S

pII

19.2

032

12.8

461

19-2

0K

9.27

9.25

0o

0.16

625

0.35

525

0.92

75

0.82

70.

577

Y0.

57S

igna

lP-n

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SS

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RA

SP

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SpI

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N0.

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090

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365

230.

911

160.

747

0.54

4N

0.57

Sig

nalP

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MS

S97

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934

38B

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++

SpI

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311

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K0.

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373

210.

756

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595

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8N

0.57

Sig

nalP

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MS

S∞

S19

7761

9228

31B

B_K

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pI5.

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3932

221

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6.08

60

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220.

5922

0.94

61

0.82

60.

701

Y0.

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igna

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SS

39.9

1524

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pII

13.8

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14.0

453

19-2

0K

0.07

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0o

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825

0.29

210.

857

20.

679

0.47

3N

0.57

Sig

nalP

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MP

37

SS

10.4

182

3746

P37

75

+S

pII

17.6

345

17.8

354

17-1

8N

0.17

0.17

0o

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721

0.48

921

0.91

114

0.83

0.64

9Y

0.57

Sig

nalP

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MM

lpH

SS

∞17

19M

lp

113

–S

pII

9.64

826

9.84

917

23-2

4K

11.3

911

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630

0.25

727

0.76

321

0.51

10.

351

N0.

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igna

lP-T

ME

rpN

SS

10.8

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1128

3278

2019

Erp

A

162

iden

tical

to B

B_P

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670

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371

250.

467

250.

893

40.

680.

567

N0.

57S

igna

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Rev

AS

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928

S11

5003

9718

ndR

ev

63lo

w d

NS

AF

ratio

for S

; nat

ive

prot

ein

is re

sita

nt to

pK

!++

+S

pII

12.6

696

12.1

3219

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K7.

397.

390

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139

260.

314

260.

892

150.

777

0.53

2N

0.57

Sig

nalP

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ME

rpP

SS

38.4

773

S11

2832

7821

20E

rpA

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2+

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12.5

726

6.71

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17-1

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4.54

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323

0.61

323

0.84

30.

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677

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igna

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QS

S∞

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2832

7839

55E

rpB

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3+

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pII

16.0

992

12.6

387

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13.9

13.9

1i5

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0.58

723

0.62

723

0.81

33

0.69

30.

658

Y0.

57S

igna

lP-n

oTM

Erp

MS

S0.

6119

4S

1128

3278

4240

Erp

B

163

low

dN

SA

F ra

tio fo

r S+

+S

pII

15.7

7914

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817

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N0.

130.

130

o0.

377

230.

547

230.

926

140.

825

0.67

8Y

0.57

Sig

nalP

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MM

lpA

SS

3.16

049

1619

Mlp

11

3+

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pII

9.72

242

9.92

333

17-1

8K

2.97

2.97

0o

0.30

423

0.44

623

0.84

320

0.66

40.

548

N0.

57S

igna

lP-n

oTM

Erp

BS

S1.

0315

8S

1128

3278

4461

Erp

B

163

low

dN

SA

F ra

tio fo

r S+

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SpI

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912

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815

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030

o0.

219

220.

383

220.

852

120.

705

0.53

4N

0.57

Sig

nalP

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MP

35

SS

1.22

449

2930

P35

60

low

dN

SA

F ra

tio fo

r S+

naS

pII

13.9

058

12.6

473

17-1

8N

0.02

0.02

0o

0.29

230.

461

230.

893

140.

761

0.60

2Y

0.57

Sig

nalP

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MM

lpJ

SS

1.82

857

2421

Mlp

11

3+

naS

pII

16.0

924

16.2

933

17-1

8N

0.42

0.42

0o

0.28

721

0.49

221

0.91

114

0.84

70.

659

Y0.

57S

igna

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Mlp

DS

S1.

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316

16M

lp

113

low

dN

SA

F ra

tio fo

r S+

+S

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15.3

2515

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919

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K0.

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110

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166

240.

339

230.

832

50.

737

0.52

6N

0.57

Sig

nalP

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ME

rpY

SS

∞S

1128

3278

2527

Erp

G

164

++

+S

pII

16.4

935

16.6

944

17-1

8N

0.17

0.17

0o

0.28

721

0.48

921

0.91

214

0.83

10.

65Y

0.57

Sig

nalP

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MM

lpC

SS

6.54

545

1716

Mlp

11

3+

+S

pII

13.7

184

9.54

449

19-2

0K

2.04

2.04

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0.22

270.

405

250.

885

210.

784

0.58

4Y

0.57

Sig

nalP

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ME

rpG

SS

∞S

2586

4455

2223

Erp

G

164

++

+S

pII

5.96

399

2.81

488

24-2

5S

21.2

321

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1i1

3-35

o0.

202

330.

269

330.

588

110.

383

0.31

1N

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Sig

nalP

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MS

0.69

645

P-O

M

2251

9960

4038

Mul

tiple

OM

toTA

TPV

TV

P–

CY

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30

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190.

3119

0.70

911

0.55

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423

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P-O

MP

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0.68

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P-O

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5199

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5156

73.

0706

516

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240

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225

280.

364

240.

869

150.

706

0.52

5N

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Sig

nalP

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MP

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P-O

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0174

228

29dN

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F ra

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lose

to 2

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ut P

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10.5

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7.66

563

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0.32

320

0.91

160.

835

0.56

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Sig

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MLp

6.6

P-O

MP

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0.59

354

P-O

M90

0929

08

13+

++

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7.64

539

7.84

6325

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V10

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10.6

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143

280.

254

280.

624

0.32

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281

N0.

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igna

lP-T

MB

esA

P-IM

P-IM

0.58

876

3539

+S

pII

5.79

043

5.99

134

15-1

6D

0.02

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0o

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241

0.19

511

0.45

31

0.39

30.

268

N0.

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igna

lP-T

MP

roX

P-IM

P-IM

0.79

348

3332

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3.75

044

3.95

135

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0K

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0o

0.13

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0.23

922

0.59

11

0.40

40.

316

N0.

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igna

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P-IM

P-IM

2.21

951

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AF

ratio

clo

se to

2.0

but

P-IM

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11.0

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921

0.36

821

0.88

11

0.77

30.

558

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igna

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Pst

SP

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931

30+

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3.91

814

4.11

905

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3T

1.03

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829

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111

0.75

73

0.63

70.

432

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igna

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P-IM

0.56

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P-O

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3982

1127

26lik

ely

mis

loca

lized

pre

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sly

(Shi

yong

)–

CY

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185

110.

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3P

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2626

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12.3

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920

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K4.

274.

270

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259

230.

418

230.

874

190.

719

0.55

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Sig

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MP

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1975

4308

4414

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31

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2529

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787

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Opp

A

37+

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6.23

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250.

219

250.

558

30.

359

0.27

1N

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Sig

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Opp

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P-IM

P-IM

0.93

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7161

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++

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527

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620

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300.

457

300.

935

210.

818

0.62

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Sig

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9202

762

57O

ppA

37

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14.3

539

14.5

548

21-2

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2.83

2.76

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0.16

622

0.36

722

0.91

24

0.83

0.58

5Y

0.57

Sig

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MIp

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P-IM

P-IM

0.56

775

P-IM

1746

4050

2221

++

++

SpI

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7410

514

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F0.

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490

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151

200.

201

200.

384

10.

30.

237

N0.

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igna

lP-T

MB

mpB

P-IM

P-IM

1.16

411

S18

1665

8538

34B

mp

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ely

mis

loca

lized

pre

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onl

y do

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y IF

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t pro

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9455

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180

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172

170.

354

170.

927

10.

764

0.54

7N

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Sig

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P-IM

P-IM

0.44

538

4039

Bm

p 36

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0.75

525

2.78

2.64

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0.20

647

0.37

538

0.15

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188

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P-IM

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619

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260.

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0.87

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P-IM

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624

0.75

21

0.50

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Sig

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MP

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824

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i13-

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0.13

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0.20

935

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MP

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7.49

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0.52

422

0.93

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Sig

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3.72

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Sig

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Sec

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by H

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ms

to b

e P

by

Mud

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; sup

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T-0

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1559

522

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22.2

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i7-2

9o0.

105

370.

139

110.

295

10.

185

0.15

6N

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Sig

nalP

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P-IM

P-IM

0.69

831

2628

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pII

2.28

024

2.48

115

21-2

2L

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0.88

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6N

0.57

Sig

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-IMP

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300.

286

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Sig

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27–

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816

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240.

345

240.

766

170.

579

0.45

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Sig

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1744

1917

likel

y m

islo

caliz

ed p

revi

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y by

IFA

, but

look

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e it'

s re

sist

ant t

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317

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K0.

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115

210.

196

210.

442

0.35

0.25

3N

0.51

Sig

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P-IM

P-IM

1.83

333

P19

8226

4849

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418

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470

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240.

558

240.

827

190.

705

0.62

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Sig

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MO

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VP

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Opp

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156

260.

199

260.

453

60.

305

0.23

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Sig

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Opp

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P-IM

P-IM

0.61

077

P-IM

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20.

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0.25

3N

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Sig

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P-IM

1.24

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P-O

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3982

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21hi

gh d

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AF

for P

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Sign

alP4

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TMH

MM

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(http

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ww

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serv

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Sign

alP4

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in v

ivo

Diff

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Tabl

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SpI

I8.

1069

38.

3078

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V6.

296.

080

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1427

0.30

827

0.93

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769

0.52

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Sig

nalP

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6733

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3.06

746

3.26

837

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1.32

1.29

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0.15

190.

324

190.

854

150.

682

0.49

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0.57

Sig

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pII

4.22

313.

4612

420

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A15

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14.1

61

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294

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374

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0.41

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9.30

329

9.50

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325

320.

316

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MS

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4.50

329

4.70

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D0.

180.

180

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129

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269

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733

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515

0.38

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Sig

nalP

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MS

pII

5.82

374.

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815

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Sig

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pII

8.21

198

4.46

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128

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Sig

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1.09

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2988

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211

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535

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Sig

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8963

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490

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Sig

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0091

0.79

1120

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101

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104

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Sig

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4.37

766

4.57

857

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047

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6.09

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0.18

118

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1379

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271

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518

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Sig

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22.4

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660.

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181

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9121

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0.79

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796

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9118

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0.15

837

0.13

911

0.26

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191

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Sig

nalP

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TMH

1.64

252

1.84

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4.87

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0.40

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0.89

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108

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135

180.

116

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Sig

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8.56

896

8.76

987

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315

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Sig

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Sig

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0.58

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865

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Sig

nalP

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pI2.

3527

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294

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399

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Sig

nalP

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0091

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437

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311

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9118

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928

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Sig

nalP

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5.29

273

4.51

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41

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194

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493

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376

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Sig

nalP

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2.17

122

2.37

213

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247.

240

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592

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441

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Sig

nalP

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2.55

115

2.75

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1.14

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47

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189

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329

200.

801

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618

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Sig

nalP

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0.86

999

1.07

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120

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1-33

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222

270.

256

270.

395

250.

234

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Sig

nalP

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0091

2.54

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20.5

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0.13

847

0.16

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0.28

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161

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9121

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9119

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600.

116

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Sig

nalP

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9.34

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9.54

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497

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616

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Sig

nalP

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9119

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1120

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816

0.49

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9116

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552

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333

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Sig

nalP

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420

0.72

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567

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101

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092

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117

200.

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0.08

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Sig

nalP

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5.36

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5.56

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945

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70

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579

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Sig

nalP

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6.89

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7.09

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324

0.76

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0.83

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3.89

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9122

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Sig

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0.91

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7.06

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9122

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Sig

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Sig

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9119

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0.22

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261

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310

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Sig

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321

400.

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214

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Sig

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1.06

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Sig

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Sig

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Sig

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270.

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Sig

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350

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Sig

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pI5.

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829

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300.

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Sig

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pI3.

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54.

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620

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218

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1i7

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0.46

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0.45

521

0.61

50.

474

0.46

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0.51

Sig

nalP

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SpI

0.99

128

1.19

219

42-4

322

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21.8

41

i20-

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0.13

243

0.17

543

0.32

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171

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9116

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1130

0.14

140.

263

90.

203

0.16

4N

0.51

Sig

nalP

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0091

18.3

118

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1o4

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0.14

125

0.13

725

0.26

71

0.14

0.13

8N

0.51

Sig

nalP

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CYT

-0.2

0091

14.3

514

.31

1i9

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0.15

425

0.11

425

0.11

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0.09

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106

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igna

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MS

pI8.

3317

68.

5326

721

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27.9

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0.41

222

0.37

222

0.56

76

0.41

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387

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igna

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MC

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912.

1015

620

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20.5

81

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0.14

149

0.15

110.

261

10.

236

0.18

2N

0.51

Sig

nalP

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SpI

6.80

286

4.32

924

25-2

69.

889.

880

o0.

381

230.

604

230.

975

200.

954

0.76

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0.57

Sig

nalP

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MS

pI5.

7124

53.

7878

625

-26

12.7

712

.77

1i7

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0.42

323

0.63

723

0.98

200.

953

0.78

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0.57

Sig

nalP

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MS

pI6.

7393

44.

3257

425

-26

10.2

910

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0o

0.38

123

0.60

423

0.97

620

0.95

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769

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TMH

0.64

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0.84

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177.

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118

110.

191

20.

143

0.12

7N

0.51

Sig

nalP

-TM

TMH

2.01

493

2.21

584

22.5

622

.54

1i2

5-47

o0.

122

530.

115

110.

186

20.

131

0.12

1N

0.51

Sig

nalP

-TM

TMH

0.68

060.

8815

120

.13

20.1

31

i12-

29o

0.16

529

0.15

229

0.27

228

0.15

90.

155

N0.

51S

igna

lP-T

MTM

H16

.059

916

.260

843

.33

24.4

62

i9-3

1o57

-70.

141

310.

107

310.

147

10.

068

0.09

3N

0.51

Sig

nalP

-TM

TMH

0.66

980.

8707

223

.02

22.3

31

i13-

35o

0.24

547

0.17

447

0.26

15

0.14

90.

165

N0.

51S

igna

lP-T

MTM

H5.

6367

55.

8376

620

.04

20.0

21

i21-

40o

0.11

410.

118

200.

214

170.

135

0.12

4N

0.51

Sig

nalP

-TM

TMH

0.00

889

0.20

9828

0.52

21.7

513

i7-2

9o33

8-0.

187

300.

138

300.

254

20.

123

0.13

3N

0.51

Sig

nalP

-TM

TMH

0.47

80.

6789

123

231

i13-

35o

0.38

131

0.12

931

0.11

45

0.05

60.

102

N0.

51S

igna

lP-T

MTM

H13

.169

910

.546

613

3.02

35.4

26

i13-

35o5

0-0.

1329

0.15

741

0.26

528

0.18

50.

167

N0.

51S

igna

lP-T

MTM

H20

.602

320

.803

298

.11

43.6

94

o10-

39i4

6-0.

151

260.

149

260.

238

140.

154

0.15

N0.

51S

igna

lP-T

MTM

H1.

5431

61.

7440

722

.85

22.8

51

i12-

34o

0.11

370.

165

110.

382

0.27

0.20

4N

0.51

Sig

nalP

-TM

TMH

13.6

3913

.839

942

.92

42.8

12

i13-

35o4

0-0.

118

260.

124

110.

183

10.

159

0.13

7N

0.51

Sig

nalP

-TM

TMH

3.78

933

3.99

024

160.

537

.18

i21-

42o4

6-0.

148

430.

087

110.

101

670.

076

0.08

3N

0.51

Sig

nalP

-TM

TMH

1.51

692

1.71

783

22.6

922

.69

1o3

0-52

i0.

106

360.

101

640.

117

560.

067

0.08

8N

0.51

Sig

nalP

-TM

TMH

0.05

040.

2513

120

.29

20.1

91

o25-

44i

0.10

641

0.09

512

0.11

811

0.09

10.

094

N0.

51S

igna

lP-T

MTM

H11

.725

411

.926

385

.89

42.5

4i7

-29o

34-5

0.14

349

0.12

949

0.18

78

0.11

0.12

2N

0.51

Sig

nalP

-TM

TMH

12.5

761

12.7

7722

.74

22.7

31

i13-

35o

0.10

535

0.13

211

0.31

51

0.15

90.

142

N0.

51S

igna

lP-T

MTM

H7.

3138

55.

5074

113

3.38

21.9

76

i9-3

1o10

0-0.

123

330.

181

330.

516

280.

191

0.18

4N

0.51

Sig

nalP

-TM

TMH

20.5

694

20.7

703

139.

1734

.85

6i7

-29o

49-7

0.14

331

0.11

811

0.19

11

0.13

50.

124

N0.

51S

igna

lP-T

MTM

H7.

0338

17.

2347

214

1.82

27.2

66

i40-

62o8

4-0.

103

560.

1323

0.21

912

0.15

50.

139

N0.

51S

igna

lP-T

MTM

H1.

9546

52.

1555

613

2.01

26.1

65

o15-

34i5

5-0.

1146

0.10

946

0.14

237

0.06

60.

093

N0.

51S

igna

lP-T

MTM

H7.

1959

27.

3968

320

2.34

43.0

49

i7-2

6o36

-50.

407

330.

144

330.

224

30.

084

0.12

1N

0.51

Sig

nalP

-TM

TMH

0.72

801

0.92

892

81.2

519

.92

4i7

-24o

72-9

0.13

331

0.13

111

0.22

63

0.17

90.

149

N0.

51S

igna

lP-T

MTM

H3.

9893

64.

1902

721

1.63

21.0

810

i20-

39o7

1-0.

1170

0.10

912

0.15

27

0.12

40.

115

N0.

51S

igna

lP-T

MTM

H1.

1216

51.

3225

621

.14

21.0

91

i7-2

6o0.

105

280.

113

110.

216

20.

120.

116

N0.

51S

igna

lP-T

MTM

H7.

5965

57.

7974

614

7.63

307

i21-

38o4

8 -0.

284

390.

307

390.

464

350.

197

0.26

6N

0.51

Sig

nalP

-TM

TMH

0.67

129

0.87

2268

.86

32.2

73

o15-

32i4

4-0.

191

330.

187

330.

241

280.

169

0.18

N0.

51S

igna

lP-T

MTM

H3.

5444

63.

7453

746

.44

21.7

72

o17-

39i3

20.

145

370.

123

130.

197

90.

157

0.13

6N

0.51

Sig

nalP

-TM

TMH

8.66

899

7.79

124

45.9

122

.57

2i9

-31o

436-

0.13

827

0.12

110.

208

20.

143

0.12

9N

0.51

Sig

nalP

-TM

TMH

18.8

917

19.0

926

53.0

437

.97

3i7

-24o

28- 4

0.19

224

0.10

711

0.17

71

0.11

0.10

8N

0.51

Sig

nalP

-TM

TMH

5.91

255

6.11

346

280.

8243

.113

o4-2

3i28

-40.

118

300.

096

110.

148

30.

092

0.09

5N

0.51

Sig

nalP

-TM

TMH

11.3

557

11.5

566

232.

3827

.44

10o1

5-37

i58-

0.13

737

0.13

737

0.22

57

0.15

60.

144

N0.

51S

igna

lP-T

MTM

H0.

3093

20.

5102

320

.59

20.4

51

o20-

39i

0.12

845

0.13

811

0.24

43

0.19

70.

16N

0.51

Sig

nalP

-TM

TMH

5.30

133

5.50

224

130.

7621

.82

6i1

3-35

o13

0.12

234

0.15

110.

283

50.

242

0.18

4N

0.51

Sig

nalP

-TM

TMH

1.53

841.

7393

111

9.22

41.5

56

i5-2

2o37

- 50.

104

220.

103

110.

143

30.

107

0.10

4N

0.51

Sig

nalP

-TM

TMH

10.0

546

10.2

555

44.1

723

.36

2o1

0-32

i354

0.13

300.

106

670.

191

0.08

70.

099

N0.

51S

igna

lP-T

MTM

H10

.517

15.

6467

353

.423

.62

2o1

0-32

i322

0.15

929

0.14

711

0.33

32

0.21

40.

172

N0.

51S

igna

lP-T

MTM

H2.

5221

32.

7230

421

.97

21.7

81

i9-3

1o0.

263

330.

187

330.

246

310.

106

0.15

7N

0.51

Sig

nalP

-TM

TMH

1.99

117

2.19

208

199.

8242

.25

9o4

-26i

38-5

0.11

127

0.11

238

0.16

334

0.08

0.1

N0.

51S

igna

lP-T

MTM

H9.

6323

9.83

321

135.

7727

.49

6i2

6-48

o58 -

0.10

526

0.09

511

0.12

665

0.09

60.

096

N0.

51S

igna

lP-T

M

8

136

Dowde

ll_TableS2

Loca

tion

Scor

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argi

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leav

age

Pos+

2Ex

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Firs

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Pred

Hel

Topo

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Ymax

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Smax

pos

Smea

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Sign

alP4

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Net

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Prot

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Ref

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for P

rev.

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Pred

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d M

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(kD

a)

Obs

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d M

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(kD

a)

Para

logo

us

Fam

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(Cas

jens

20

00)

Num

ber

of

Para

logs

Com

men

tsIm

mun

oge

nici

tyTn

m

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t

Lipo

P1.0

(http

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ww

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serv

ices

/Lip

oP/)

TMH

MM

2.0

(http

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ww

.cbs

.dtu

.dk/

serv

ices

/TM

HM

M-2

.0/)

Sign

alP4

.1 g

ram

- pre

dict

ions

(h

ttp://

ww

w.c

bs.d

tu.d

k/se

rvic

es/S

igna

lP/)

in v

ivo

Diff

eren

tial E

xpre

ssio

n

Tabl

e 1

TMH

0.00747

0.20838

19.35

19.34

1i5-23o

0.127

290.102

110.194

10.092

0.098N

0.51

SignalP-TM

TMH

10.6611

6.06579

52.77

22.23

2i21-43o11

0.113

440.174

220.431

190.27

0.21

N0.51

SignalP-TM

TMH

5.96773

5.1117

47.48

21.94

1i7-29o

0.153

210.227

210.582

20.39

0.287N

0.51

SignalP-TM

TMH

5.22203

5.42294

251.25

22.65

12i13-35o83-

0.119

380.176

110.381

40.323

0.23

N0.51

SignalP-TM

TMH

1.07298

1.27389

276.04

33.75

12i23-45o50-

0.108

390.112

700.157

650.101

0.108N

0.51

SignalP-TM

TMH

3.32153

3.52244

43.66

40.6

2o5-27i40-6

0.119

280.112

400.153

300.104

0.11

N0.51

SignalP-TM

TMH

6.10431

5.07437

214.47

22.88

9o20-42i75-

0.174

430.168

430.243

160.157

0.164N

0.51

SignalP-TM

TMH

2.28605

2.48696

42.85

35.45

2o15-34i47-

0.102

640.122

110.176

20.155

0.134N

0.51

SignalP-TM

TMH

6.14452

4.95182

90.87

22.02

4i19-41o27

0.134

330.141

180.232

80.194

0.161N

0.51

SignalP-TM

TMH

2.20357

2.40448

20.11

20.1

1o15-34i

0.134

320.124

320.186

300.108

0.118N

0.51

SignalP-TM

CYT

-0.20091

256.62

20.63

12i7-29o206-

0.107

370.091

110.1

40.088

0.09

N0.51

SignalP-TM

CYT

-0.20091

168.7

09o308-325i

0.106

180.141

180.23

10.178

0.158N

0.57

SignalP-noTM

CYT

-0.20091

192.73

09o297-319i

0.109

200.102

360.12

270.098

0.1N

0.57

SignalP-noTM

CYT

-0.20091

190.93

09o298-320i

0.124

200.146

200.259

20.185

0.164N

0.57

SignalP-noTM

CYT

-0.20091

0.57107

200.29

22.49

9i33-55o79-

0.102

640.103

110.132

10.109

0.106N

0.51

SignalP-TM

CYT

-0.20091

157.63

1.36

7i145-167o

0.191

230.31

230.667

170.502

0.4N

0.57

SignalP-noTM

CYT

-0.20091

138.65

22.06

7i28-50o15

0.106

440.107

170.139

90.112

0.109N

0.51

SignalP-TM

CYT

-0.20091

131.13

19.83

6i42-64o68-

0.112

250.086

250.107

700.07

0.08

N0.51

SignalP-TM

CYT

-0.20091

0.00782

117.61

22.62

5i7-29o102-

0.122

300.097

610.108

10.066

0.085N

0.51

SignalP-TM

CYT

-0.20091

109.89

21.45

5i16-38o40

0.113

300.13

110.289

290.177

0.147N

0.51

SignalP-TM

CYT

-0.20091

1.46371

124.08

20.91

5i7-24o339-

0.116

300.167

110.355

20.284

0.21

N0.51

SignalP-TM

CYT

-0.20091

2.00927

81.11

33.92

4o4-26i47-6

0.113

210.109

110.178

30.119

0.112N

0.51

SignalP-TM

CYT

-0.20091

101.37

04i122-144o

0.102

700.105

150.139

60.108

0.106N

0.57

SignalP-noTM

CYT

-0.20091

94.05

04i282-299o

0.11

560.104

560.127

520.083

0.094N

0.57

SignalP-noTM

CYT

-0.20091

87.85

04o265-287i2

0.121

450.11

450.128

350.098

0.105N

0.57

SignalP-noTM

CYT

-0.20091

81.76

37.33

4i7-24o29-5

0.106

260.096

690.111

60.061

0.083N

0.51

SignalP-TM

CYT

-0.20091

63.48

22.4

3o15-36i78-

0.099

450.092

620.092

560.056

0.078N

0.51

SignalP-TM

CYT

-0.20091

2.58781

93.47

23.15

3o28-50i62-

0.164

270.164

270.239

160.169

0.166N

0.51

SignalP-TM

CYT

-0.20091

0.75566

76.09

28.39

3i45-62o67-

0.157

200.271

200.578

150.446

0.336N

0.51

SignalP-TM

CYT

-0.20091

44.73

0.06

2i381-403o4

0.147

270.253

230.605

200.438

0.34

N0.57

SignalP-noTM

CYT

-0.20091

36.01

12.27

2o43-60i15

0.133

640.123

640.181

10.115

0.119N

0.57

SignalP-noTM

CYT

-0.20091

79.82

02o331-352i

0.122

550.111

410.142

310.094

0.103N

0.57

SignalP-noTM

CYT

-0.20091

1.14429

44.59

20.81

2i25-44o472

0.128

510.139

130.199

100.183

0.155N

0.51

SignalP-TM

CYT

-0.20091

1.26635

44.01

21.41

2o15-33i30

0.128

210.138

210.245

20.166

0.148N

0.51

SignalP-TM

CYT

-0.20091

1.08125

43.44

21.58

2i7-29o473-

0.113

540.141

110.264

10.2

0.163N

0.51

SignalP-TM

CYT

-0.20091

40.22

0.4

2i85-102o12

0.127

240.148

240.288

230.166

0.156N

0.57

SignalP-noTM

CYT

-0.20091

41.59

9.35

2i52-71o76-

0.106

690.124

510.187

420.124

0.124N

0.51

SignalP-TM

CYT

-0.20091

13.91

10.34

1i44-66o

0.124

220.105

430.138

610.094

0.101N

0.51

SignalP-TM

CYT

-0.20091

2.42188

42.7

5.15

1i138-160o

0.168

190.304

190.796

10.546

0.418N

0.57

SignalP-noTM

CYT

-0.20091

22.2

01o285-307i

0.111

480.106

210.134

150.104

0.105N

0.57

SignalP-noTM

CYT

-0.20091

23.03

01i329-351o

0.101

430.102

660.115

570.089

0.096N

0.57

SignalP-noTM

CYT

-0.20091

22.6

01i121-143o

0.167

560.121

560.142

80.098

0.11

N0.57

SignalP-noTM

CYT

-0.20091

27.68

01i330-352o

0.104

260.102

300.143

20.094

0.098N

0.57

SignalP-noTM

CYT

-0.20091

19.72

01o246-268i

0.105

390.138

220.235

110.162

0.149N

0.57

SignalP-noTM

CYT

-0.20091

19.95

01i92-111o

0.143

380.117

380.13

350.094

0.106N

0.57

SignalP-noTM

CYT

-0.20091

22.64

01i239-261o

0.139

300.12

300.164

70.111

0.116N

0.57

SignalP-noTM

CYT

-0.20091

23.15

01o193-215i

0.155

300.127

300.134

70.102

0.115N

0.57

SignalP-noTM

CYT

-0.20091

22.32

01o160-182i

0.115

430.11

590.143

510.101

0.105N

0.57

SignalP-noTM

CYT

-0.20091

22.69

01o171-193i

0.122

410.121

200.195

10.132

0.126N

0.57

SignalP-noTM

CYT

-0.20091

21.55

01o165-184i

0.101

270.106

110.143

50.11

0.108N

0.57

SignalP-noTM

CYT

-0.20091

20.25

01i197-216o

0.104

280.118

110.171

10.137

0.127N

0.57

SignalP-noTM

CYT

-0.20091

22.69

01o171-193i

0.122

410.121

200.195

10.132

0.126N

0.57

SignalP-noTM

CYT

-0.20091

19.78

01i154-173o

0.102

280.106

400.127

20.098

0.102N

0.57

SignalP-noTM

CYT

-0.20091

22.69

01o182-204i

0.122

410.121

200.195

10.132

0.126N

0.57

SignalP-noTM

CYT

-0.20091

20.18

01i165-184o

0.103

280.12

110.181

50.141

0.13

N0.57

SignalP-noTM

CYT

-0.20091

21.58

01i183-205o

0.105

400.118

110.171

20.136

0.126N

0.57

SignalP-noTM

CYT

-0.20091

22.68

01o215-237i

0.124

380.112

380.131

120.101

0.107N

0.57

SignalP-noTM

CYT

-0.20091

22.7

01o153-175i

0.128

200.124

200.191

10.129

0.126N

0.57

SignalP-noTM

CYT

-0.20091

22.68

01o186-208i

0.124

380.113

380.133

120.103

0.108N

0.57

SignalP-noTM

CYT

-0.20091

21.17

01i169-188o

0.101

280.131

110.217

10.167

0.148N

0.57

SignalP-noTM

CYT

-0.20091

10.57

9.24

0o

0.162

220.225

220.479

210.302

0.253N

0.51

SignalP-TM

CYT

-0.20091

3.46

0.59

0o

0.109

690.11

110.159

20.12

0.114N

0.57

SignalP-noTM

CYT

-0.20091

0.39

00o

0.107

300.136

110.227

20.185

0.159N

0.57

SignalP-noTM

CYT

-0.20091

0.09

0.05

0o

0.121

290.111

510.134

390.1

0.106N

0.57

SignalP-noTM

CYT

-0.20091

13.16

13.15

0o

0.111

370.107

460.142

360.085

0.099N

0.51

SignalP-TM

CYT

-0.20091

0.66

0.12

0o

0.114

360.122

110.208

10.145

0.133N

0.57

SignalP-noTM

CYT

-0.20091

3.55

00o

0.103

220.123

150.204

120.144

0.133N

0.57

SignalP-noTM

CYT

-0.20091

0.92

00o

0.104

340.123

170.176

110.152

0.137N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.108

180.107

300.129

270.101

0.104N

0.57

SignalP-noTM

CYT

-0.20091

2.22

2.22

0o

0.314

370.263

370.351

320.178

0.223N

0.57

SignalP-noTM

CYT

-0.20091

0.09

00o

0.101

540.114

110.164

10.13

0.121N

0.57

SignalP-noTM

CYT

-0.20091

3.01

0.01

0o

0.122

210.123

210.156

120.117

0.121N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.11

210.111

210.141

140.11

0.111N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.101

370.103

680.117

620.088

0.096N

0.57

SignalP-noTM

CYT

-0.20091

2.68

00o

0.117

220.183

220.426

180.282

0.23

N0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.139

390.125

110.201

20.155

0.139N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.26

200.219

200.263

30.205

0.212N

0.57

SignalP-noTM

CYT

-0.20091

0.1

00o

0.102

200.133

110.223

10.176

0.153N

0.57

SignalP-noTM

CYT

-0.20091

0.07

00o

0.12

400.111

110.164

30.129

0.12

N0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.13

600.116

600.115

520.091

0.104N

0.57

SignalP-noTM

CYT

-0.20091

0.05

0.01

0o

0.107

460.101

590.113

500.09

0.096N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.13

130.153

130.289

120.18

0.166N

0.57

SignalP-noTM

CYT

-0.20091

2.76

2.17

0o

0.107

530.106

530.143

510.104

0.105N

0.57

SignalP-noTM

CYT

-0.20091

0.42

0.42

0o

0.141

250.15

250.214

220.152

0.151N

0.57

SignalP-noTM

CYT

-0.20091

33.83

7.17

0o

0.207

220.294

220.708

40.489

0.386N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.1

390.103

390.12

340.091

0.097N

0.57

SignalP-noTM

CYT

-0.20091

0.4

00o

0.1

580.12

110.178

30.146

0.132N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.101

380.108

510.129

440.094

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.102

200.104

200.135

100.1

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.75089

3.03

1.81

0o

0.179

200.347

200.868

10.705

0.515N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.102

400.102

580.117

100.094

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.06

0.05

0o

0.171

410.144

110.271

30.203

0.172N

0.57

SignalP-noTM

CYT

-0.20091

0.79

00o

0.11

290.106

120.132

60.113

0.109N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.11

250.119

250.147

140.127

0.123N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

590.097

700.108

440.088

0.093N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.111

210.126

110.214

60.167

0.146N

0.57

SignalP-noTM

CYT

-0.20091

0.84

0.83

0o

0.125

620.128

110.225

20.156

0.141N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.01

0o

0.106

240.196

200.519

120.324

0.256N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.118

250.107

250.132

240.08

0.094N

0.57

SignalP-noTM

CYT

-0.20091

8.56

0.02

0o

0.131

200.121

200.171

60.126

0.123N

0.57

SignalP-noTM

CYT

-0.20091

1.6882

22.99

11.46

0o

0.179

210.211

210.345

180.244

0.223N

0.51

SignalP-TM

CYT

-0.20091

0.21

00o

0.128

210.174

130.405

90.281

0.224N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.104

450.102

250.12

420.091

0.097N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.101

580.121

180.172

90.139

0.129N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.104

430.104

240.127

130.093

0.099N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.101

210.116

530.174

430.104

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.3

00o

0.1

590.099

210.127

110.095

0.097N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.135

180.127

180.165

120.112

0.12

N0.57

SignalP-noTM

CYT

-0.20091

1.3

0.08

0o

0.275

430.18

430.238

420.108

0.146N

0.57

SignalP-noTM

CYT

-0.20091

0.06

0.05

0o

0.108

450.136

500.259

440.117

0.127N

0.57

SignalP-noTM

CYT

-0.20091

0.55

0.16

0o

0.107

320.111

320.128

210.105

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.1

00o

0.101

370.099

110.114

10.097

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.06

00o

0.118

310.106

310.117

490.089

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.11

550.103

680.116

90.092

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.114

370.125

370.187

330.114

0.12

N0.57

SignalP-noTM

CYT

-0.20091

0.14

0.04

0o

0.107

220.111

220.134

10.114

0.112N

0.57

SignalP-noTM

9

137

Dowde

ll_TableS2

Loca

tion

Scor

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Pos+

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Prot

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Loc.

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Ref

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for P

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Pred

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(kD

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Obs

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Para

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Fam

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(Cas

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Num

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of

Para

logs

Com

men

tsIm

mun

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nici

tyTn

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Lipo

P1.0

(http

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serv

ices

/Lip

oP/)

TMH

MM

2.0

(http

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serv

ices

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Sign

alP4

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- pre

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(h

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in v

ivo

Diff

eren

tial E

xpre

ssio

n

Tabl

e 1

CYT

-0.20091

0.03

00o

0.209

550.134

110.231

40.182

0.157N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.125

630.106

630.127

90.092

0.1N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.113

150.108

150.139

10.108

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.16

00o

0.11

240.102

240.157

10.089

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.106

360.106

250.14

180.1

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.111

310.106

310.138

220.097

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.1

680.101

390.117

230.091

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.02

0o

0.113

220.111

220.123

200.101

0.107N

0.57

SignalP-noTM

CYT

-0.20091

0.27

0.24

0o

0.199

200.132

200.157

390.081

0.108N

0.57

SignalP-noTM

CYT

-0.20091

2.45359

1.23

1.19

0o

0.131

320.162

320.321

300.203

0.181N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.105

310.104

420.118

380.096

0.1N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.107

330.107

480.14

10.101

0.104N

0.57

SignalP-noTM

CYT

-0.20091

0.12

00o

0.114

530.11

200.145

120.108

0.109N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.103

580.122

460.225

400.098

0.111N

0.57

SignalP-noTM

CYT

-0.20091

1.34

0.02

0o

0.14

290.184

290.353

230.216

0.199N

0.57

SignalP-noTM

CYT

-0.20091

0.05

0.03

0o

0.114

240.108

240.124

230.095

0.102N

0.57

SignalP-noTM

CYT

-0.20091

2.61

0.13

0i

0.152

360.158

620.366

40.156

0.157N

0.57

SignalP-noTM

CYT

-0.20091

0.12

00i

0.1

570.114

110.148

10.132

0.123N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.106

250.099

690.12

490.088

0.094N

0.57

SignalP-noTM

CYT

-0.20091

0.56

00o

0.102

200.104

590.121

480.087

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.104

200.101

290.116

240.089

0.095N

0.57

SignalP-noTM

CYT

-0.20091

0.14

0.01

0o

0.116

390.124

390.173

320.115

0.12

N0.57

SignalP-noTM

CYT

-0.20091

0.29

0.04

0o

0.121

350.123

530.223

450.103

0.114N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.105

200.119

200.167

100.135

0.127N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.16

0o

0.106

310.122

460.174

500.131

0.126N

0.57

SignalP-noTM

CYT

-0.20091

0.15

0.02

0o

0.104

250.114

120.149

20.133

0.123N

0.57

SignalP-noTM

CYT

-0.20091

3.33

3.26

0o

0.103

490.115

540.162

480.111

0.113N

0.57

SignalP-noTM

CYT

-0.20091

0.06

00o

0.103

210.098

670.113

80.092

0.095N

0.57

SignalP-noTM

CYT

-0.20091

3.25

00o

0.123

460.107

200.166

10.11

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.111

320.131

110.21

20.171

0.15

N0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.105

500.108

280.133

230.101

0.105N

0.57

SignalP-noTM

CYT

-0.20091

0.05

0.01

0o

0.103

590.11

110.159

50.128

0.119N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

520.117

120.15

90.136

0.126N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.116

210.117

210.143

270.108

0.113N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.208

310.196

310.277

70.205

0.2N

0.57

SignalP-noTM

CYT

-0.20091

0.35

0.35

0o

0.112

570.106

220.122

250.1

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.59

00o

0.158

190.141

190.181

20.137

0.139N

0.57

SignalP-noTM

CYT

-0.20091

3.37

2.45

0o

0.098

450.101

130.124

30.107

0.103N

0.51

SignalP-TM

CYT

-0.20091

5.46

00o

0.104

210.103

320.141

30.095

0.099N

0.57

SignalP-noTM

CYT

-0.20091

0.35

00o

0.113

490.106

280.125

210.103

0.104N

0.57

SignalP-noTM

CYT

-0.20091

4.21

2.59

0o

0.133

440.131

440.167

500.111

0.122N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.01

0o

0.129

210.149

210.235

180.185

0.166N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

250.101

340.112

270.083

0.092N

0.57

SignalP-noTM

CYT

-0.20091

1.46

0.09

0o

0.107

490.104

110.145

10.108

0.106N

0.57

SignalP-noTM

CYT

-0.20091

0.26

0.01

0o

0.107

390.12

390.183

40.134

0.126N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

100.108

430.124

370.085

0.097N

0.57

SignalP-noTM

CYT

-0.20091

0.08

0.01

0o

0.11

430.112

220.181

390.118

0.115N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.127

240.105

240.12

570.087

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.128

390.126

390.158

290.103

0.115N

0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.107

360.102

570.129

530.09

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.2

0.18

0o

0.261

210.203

210.342

480.173

0.189N

0.57

SignalP-noTM

CYT

-0.20091

1.59468

2.42

0.04

0o

0.133

410.129

260.2

210.153

0.14

N0.57

SignalP-noTM

CYT

-0.20091

0.8

00o

0.199

640.137

640.187

190.115

0.127N

0.57

SignalP-noTM

CYT

-0.20091

2.91

00o

0.101

410.106

410.139

10.105

0.105N

0.57

SignalP-noTM

CYT

-0.20091

0.51

00o

0.119

270.115

110.229

10.119

0.117N

0.57

SignalP-noTM

CYT

-0.20091

1.94

0.49

0o

0.156

350.146

350.186

340.131

0.139N

0.57

SignalP-noTM

CYT

-0.20091

0.16

0.07

0o

0.137

470.121

470.148

20.099

0.11

N0.57

SignalP-noTM

CYT

-0.20091

1.24

0.01

0o

0.103

290.134

120.247

80.186

0.158N

0.57

SignalP-noTM

CYT

-0.20091

0.69

00o

0.103

470.1

690.107

610.092

0.096N

0.57

SignalP-noTM

CYT

-0.20091

0.11

0.11

0o

0.119

170.197

170.409

30.341

0.25

N0.51

SignalP-TM

CYT

-0.20091

1.63

00o

0.131

400.122

400.133

390.097

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.137

560.121

110.163

10.148

0.134N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.115

360.126

220.186

70.148

0.136N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.12

500.107

250.114

220.097

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.15

0.02

0i

0.113

240.155

110.334

20.239

0.195N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.117

490.118

110.192

30.136

0.126N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.139

240.132

530.199

370.139

0.135N

0.57

SignalP-noTM

CYT

-0.20091

0.17

00o

0.102

250.101

700.139

240.097

0.099N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.129

230.113

230.156

10.101

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0i

0.106

670.128

140.189

520.165

0.145N

0.57

SignalP-noTM

CYT

-0.20091

1.33

00o

0.128

180.165

180.367

170.21

0.186N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.105

160.12

160.162

100.135

0.127N

0.57

SignalP-noTM

CYT

-0.20091

12.28

0.02

0o

0.136

280.14

280.264

70.162

0.151N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.122

470.123

470.159

340.104

0.114N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.122

430.111

430.144

120.105

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.102

470.105

380.142

270.092

0.099N

0.57

SignalP-noTM

CYT

-0.20091

5.7

5.59

0o

0.129

250.204

110.595

10.388

0.29

N0.57

SignalP-noTM

CYT

-0.20091

0.75

0.74

0o

0.112

360.127

110.222

50.17

0.147N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.135

260.111

260.146

320.101

0.106N

0.57

SignalP-noTM

CYT

-0.20091

0.5

0.49

0o

0.125

310.163

110.312

60.277

0.216N

0.57

SignalP-noTM

CYT

-0.20091

0.82

00o

0.161

320.126

320.137

20.101

0.114N

0.57

SignalP-noTM

CYT

-0.20091

0.14

0.01

0o

0.143

590.117

590.161

70.102

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.02

0.01

0o

0.128

600.112

600.113

530.085

0.099N

0.57

SignalP-noTM

CYT

-0.20091

17.32

00o

0.108

280.105

140.122

110.111

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.115

520.165

160.342

80.272

0.215N

0.57

SignalP-noTM

CYT

-0.20091

0.12

00o

0.103

270.11

350.136

260.102

0.106N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

290.1

340.112

320.084

0.092N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.17

0o

0.161

390.159

510.276

460.109

0.136N

0.57

SignalP-noTM

CYT

-0.20091

0.11

00o

0.11

440.105

110.125

60.113

0.109N

0.57

SignalP-noTM

CYT

-0.20091

0.21

00o

0.138

370.113

370.121

360.093

0.104N

0.57

SignalP-noTM

CYT

-0.20091

0.03

0.01

0o

0.116

330.12

180.159

110.129

0.124N

0.57

SignalP-noTM

CYT

-0.20091

0.04

0.04

0o

0.271

220.403

220.722

110.582

0.487N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.06

0o

0.11

340.112

510.142

410.093

0.103N

0.57

SignalP-noTM

CYT

-0.20091

13.97

0.56

0o

0.117

480.194

130.456

90.375

0.279N

0.57

SignalP-noTM

CYT

-0.20091

3.35

00o

0.103

210.095

310.103

700.086

0.091N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.113

470.109

470.113

400.093

0.101N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.106

370.096

680.105

440.081

0.089N

0.57

SignalP-noTM

CYT

-0.20091

0.12

0.04

0o

0.11

390.155

110.365

10.226

0.189N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.137

170.13

170.205

100.116

0.124N

0.57

SignalP-noTM

CYT

-0.20091

0.08

0.03

0o

0.107

230.102

230.132

10.096

0.099N

0.57

SignalP-noTM

CYT

-0.20091

0.06

0.02

0o

0.105

360.11

410.132

350.105

0.108N

0.57

SignalP-noTM

CYT

-0.20091

2.38

0.02

0o

0.111

560.136

110.259

20.185

0.159N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.105

390.105

260.127

200.101

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.1

0.02

0o

0.111

460.099

460.104

150.09

0.095N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.107

560.1

320.13

60.095

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.22

0.01

0o

0.139

180.144

180.244

10.155

0.149N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.137

450.131

450.189

430.102

0.117N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.101

550.111

140.156

60.123

0.117N

0.57

SignalP-noTM

CYT

-0.20091

13.02

2.05

0o

0.109

630.1

630.117

490.087

0.094N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.105

290.122

110.187

40.152

0.136N

0.57

SignalP-noTM

CYT

-0.20091

0.18

0.01

0o

0.102

420.121

110.219

10.135

0.128N

0.57

SignalP-noTM

CYT

-0.20091

9.03

00o

0.1

340.106

260.131

120.103

0.105N

0.57

SignalP-noTM

CYT

-0.20091

0.59

0.33

0o

0.185

240.193

240.417

450.214

0.203N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.01

0o

0.113

100.135

130.227

90.183

0.158N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.113

300.115

110.201

30.131

0.123N

0.57

SignalP-noTM

CYT

-0.20091

0.04

0.03

0o

0.105

200.126

170.177

160.151

0.138N

0.57

SignalP-noTM

CYT

-0.20091

0.07

00o

0.104

310.11

570.139

480.108

0.109N

0.57

SignalP-noTM

10

138

Dowde

ll_TableS2

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in v

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Diff

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tial E

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n

Tabl

e 1

CYT

-0.20091

0.05

00o

0.103

210.163

110.346

10.258

0.208N

0.57

SignalP-noTM

CYT

-0.20091

0.37

0.34

0o

0.109

550.12

550.164

470.12

0.12

N0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.103

460.101

230.127

190.104

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.119

500.151

110.334

10.218

0.183N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.124

480.107

480.117

290.097

0.102N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.254

360.167

360.198

90.126

0.148N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.103

460.111

110.172

30.127

0.119N

0.57

SignalP-noTM

CYT

-0.20091

0.2

00o

0.106

180.114

110.16

20.132

0.123N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.106

180.104

500.122

400.095

0.1N

0.57

SignalP-noTM

CYT

-0.20091

0.09

00i

0.121

440.139

120.222

20.196

0.166N

0.57

SignalP-noTM

CYT

-0.20091

0.28

0.21

0o

0.123

340.149

340.247

220.162

0.155N

0.57

SignalP-noTM

CYT

-0.20091

0.08

0.08

0o

0.104

600.159

110.331

10.248

0.201N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

350.135

120.205

60.182

0.157N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.217

460.157

460.201

290.12

0.14

N0.57

SignalP-noTM

CYT

-0.20091

0.11

0.1

0o

0.117

490.13

490.206

480.126

0.128N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

610.109

610.132

430.096

0.103N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.116

330.192

160.544

120.361

0.271N

0.57

SignalP-noTM

CYT

-0.20091

0.63

0.02

0o

0.109

520.111

520.146

10.106

0.109N

0.57

SignalP-noTM

CYT

-0.20091

2.45

2.28

0o

0.331

180.349

180.429

140.361

0.354N

0.51

SignalP-TM

CYT

-0.20091

0.05

00o

0.108

280.12

190.186

180.142

0.13

N0.57

SignalP-noTM

CYT

-0.20091

2.43

00o

0.102

250.104

140.121

90.109

0.106N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.118

180.116

180.146

20.12

0.118N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.099

310.122

180.191

130.133

0.127N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.15

0o

0.109

90.112

510.144

430.103

0.107N

0.57

SignalP-noTM

CYT

-0.20091

0.2

00o

0.102

530.103

530.137

380.089

0.096N

0.57

SignalP-noTM

CYT

-0.20091

5.88

1.75

0o

0.446

220.285

220.392

190.169

0.231N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.02

0o

0.108

290.153

110.298

10.227

0.188N

0.57

SignalP-noTM

CYT

-0.20091

0.1

0.02

0o

0.124

300.166

300.386

250.202

0.183N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.101

620.134

110.248

30.178

0.155N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.104

240.121

220.188

140.133

0.127N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.15

360.115

360.144

10.099

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.108

420.104

420.107

350.089

0.097N

0.57

SignalP-noTM

CYT

-0.20091

0.12

00o

0.127

220.136

110.273

200.181

0.157N

0.57

SignalP-noTM

CYT

-0.20091

13.78

0.03

0o

0.113

270.138

160.239

140.182

0.159N

0.57

SignalP-noTM

CYT

-0.20091

0.04

0.03

0o

0.131

300.145

150.274

90.203

0.172N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.126

430.12

430.16

400.085

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.19

0.01

0o

0.109

260.145

110.324

20.202

0.172N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.127

220.121

120.165

30.146

0.133N

0.57

SignalP-noTM

CYT

-0.20091

0.34

00o

0.103

260.105

110.14

10.11

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.169

260.148

260.163

10.128

0.139N

0.57

SignalP-noTM

CYT

-0.20091

6.5

5.42

0o

0.113

590.104

400.139

10.082

0.096N

0.51

SignalP-TM

CYT

-0.20091

0.77

0.12

0o

0.141

200.176

200.332

150.196

0.185N

0.57

SignalP-noTM

CYT

-0.20091

1.51431

41.49

20.78

0o

0.222

210.327

210.736

110.52

0.418N

0.57

SignalP-noTM

CYT

-0.20091

0.05

0.03

0o

0.222

600.15

600.128

50.09

0.122N

0.57

SignalP-noTM

CYT

-0.20091

0.07

00o

0.107

700.101

370.111

330.087

0.095N

0.57

SignalP-noTM

CYT

-0.20091

0.94

0.01

0o

0.139

220.134

220.189

170.131

0.132N

0.57

SignalP-noTM

CYT

-0.20091

0.06

00o

0.122

260.098

320.128

10.075

0.087N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.156

330.128

200.16

20.123

0.126N

0.57

SignalP-noTM

CYT

-0.20091

12.98

0.02

0o

0.237

260.23

260.392

190.192

0.212N

0.57

SignalP-noTM

CYT

-0.20091

0.46

00o

0.118

330.117

330.14

200.113

0.115N

0.57

SignalP-noTM

CYT

-0.20091

0.12

00o

0.108

360.18

110.444

40.346

0.258N

0.57

SignalP-noTM

CYT

-0.20091

4.96

00o

0.127

290.163

210.321

180.267

0.212N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.107

440.104

230.122

140.095

0.1N

0.57

SignalP-noTM

CYT

-0.20091

2.83

2.83

0o

0.361

190.365

190.522

20.393

0.375N

0.51

SignalP-TM

CYT

-0.20091

14.06

11.82

0o

0.116

250.111

110.175

10.122

0.115N

0.51

SignalP-TM

CYT

-0.20091

0.48

0.47

0o

0.207

340.202

210.471

90.373

0.282N

0.57

SignalP-noTM

CYT

-0.20091

0.11

0.02

0o

0.103

300.161

200.351

120.228

0.193N

0.57

SignalP-noTM

CYT

-0.20091

0.49

00o

0.117

480.109

480.116

460.095

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.09

0o

0.119

240.149

430.349

370.155

0.152N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.101

330.105

200.122

110.107

0.106N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.104

280.104

350.145

270.101

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.92

0.81

0o

0.113

340.161

210.361

20.244

0.2N

0.57

SignalP-noTM

CYT

-0.20091

0.08

0.05

0o

0.106

570.12

110.196

30.141

0.13

N0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.104

280.158

110.37

10.235

0.194N

0.57

SignalP-noTM

CYT

-0.20091

0.22

0.02

0o

0.109

290.121

110.207

20.145

0.132N

0.57

SignalP-noTM

CYT

-0.20091

0.05

00o

0.103

310.113

420.166

400.107

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.109

520.124

130.174

10.155

0.138N

0.57

SignalP-noTM

CYT

-0.20091

6.48

0.01

0o

0.119

310.117

310.167

80.101

0.11

N0.57

SignalP-noTM

CYT

-0.20091

8.04

0.06

0o

0.14

250.203

250.457

10.332

0.263N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.12

400.114

400.156

10.103

0.108N

0.57

SignalP-noTM

CYT

-0.20091

1.31

0.02

0o

0.133

580.114

110.149

20.13

0.121N

0.57

SignalP-noTM

CYT

-0.20091

0.27

0.06

0o

0.109

400.125

220.202

30.155

0.139N

0.57

SignalP-noTM

CYT

-0.20091

1.31

1.28

0o

0.102

460.111

280.134

180.108

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.04

0.03

0o

0.168

360.177

180.36

150.241

0.207N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.109

390.106

570.115

530.089

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.108

30.107

290.174

20.11

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.117

170.131

170.234

10.155

0.143N

0.57

SignalP-noTM

CYT

-0.20091

0.03

0.02

0o

0.107

350.125

400.172

330.124

0.125N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.105

430.105

680.117

560.097

0.101N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.124

670.106

220.134

130.1

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.21

0.21

0o

0.188

390.137

390.113

360.09

0.115N

0.57

SignalP-noTM

CYT

-0.20091

0.8

00o

0.115

200.106

410.121

350.087

0.097N

0.57

SignalP-noTM

CYT

-0.20091

0.07

00o

0.111

210.114

210.13

150.107

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.43

0.27

0o

0.117

300.115

300.231

490.1

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.2

0.2

0o

0.265

200.259

200.382

190.261

0.259N

0.51

SignalP-TM

CYT

-0.20091

0.56

00o

0.123

250.125

110.195

10.155

0.139N

0.57

SignalP-noTM

CYT

-0.20091

0.75

0.64

0o

0.124

310.157

180.322

150.217

0.186N

0.57

SignalP-noTM

CYT

-0.20091

0.23

00o

0.157

360.115

360.112

110.089

0.103N

0.57

SignalP-noTM

CYT

-0.20091

4.16

0.08

0o

0.121

300.151

170.34

160.224

0.185N

0.57

SignalP-noTM

CYT

-0.20091

11.68

0.52

0o

0.149

450.117

450.113

180.093

0.105N

0.57

SignalP-noTM

CYT

-0.20091

18.5

1.03

0o

0.113

220.11

220.146

10.108

0.109N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.02

0o

0.105

460.112

110.194

10.117

0.115N

0.57

SignalP-noTM

CYT

-0.20091

2.61

00o

0.109

330.108

120.141

120.117

0.112N

0.57

SignalP-noTM

CYT

-0.20091

0.83

0.03

0o

0.137

330.189

330.384

290.209

0.199N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.136

450.118

130.16

80.132

0.124N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

220.107

320.145

690.095

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.01

0o

0.119

260.179

110.396

10.316

0.243N

0.57

SignalP-noTM

CYT

-0.20091

0.23

0.04

0o

0.112

390.106

390.127

10.099

0.103N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

530.107

690.122

650.095

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.36

00o

0.109

490.127

220.197

180.152

0.139N

0.57

SignalP-noTM

CYT

-0.20091

0.57

0.25

0o

0.115

220.188

180.432

100.32

0.237N

0.51

SignalP-TM

CYT

-0.20091

00

0o

0.101

270.102

110.121

20.105

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.099

390.104

370.127

300.095

0.1N

0.57

SignalP-noTM

CYT

-0.20091

1.71

0.39

0o

0.135

510.131

120.212

40.171

0.15

N0.57

SignalP-noTM

CYT

-0.20091

1.35

0.03

0o

0.155

370.13

370.144

330.103

0.117N

0.57

SignalP-noTM

CYT

-0.20091

0.16

0.05

0o

0.113

210.174

110.409

10.292

0.229N

0.57

SignalP-noTM

CYT

-0.20091

0.48

0.03

0o

0.123

240.133

110.212

10.175

0.153N

0.57

SignalP-noTM

CYT

-0.20091

0.08

00o

0.106

420.107

690.138

20.101

0.104N

0.57

SignalP-noTM

CYT

-0.20091

0.51

0.18

0o

0.103

620.158

110.311

20.249

0.201N

0.57

SignalP-noTM

CYT

-0.20091

0.21

0.19

0o

0.13

400.138

400.171

280.131

0.135N

0.57

SignalP-noTM

CYT

-0.20091

0.06

0.05

0o

0.119

440.252

200.669

180.569

0.401N

0.57

SignalP-noTM

CYT

-0.20091

1.74

0.1

0o

0.107

490.111

420.144

150.105

0.108N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.135

360.121

360.165

20.104

0.113N

0.57

SignalP-noTM

CYT

-0.20091

0.05

0.02

0o

0.172

210.244

210.468

170.317

0.278N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.106

240.099

430.107

410.09

0.095N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.122

270.158

110.355

20.248

0.2N

0.57

SignalP-noTM

CYT

-0.20091

0.04

00o

0.104

470.113

310.177

450.107

0.11

N0.57

SignalP-noTM

11

139

Dowde

ll_TableS2

Loca

tion

Scor

eM

argi

nC

leav

age

Pos+

2Ex

pAA

Firs

t60

Pred

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Smea

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Sign

alP4

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onse

nsu

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max

cut

Net

wor

ks-

used

Prot

ein

Nam

eC

onse

ns. L

oc.

His

-tag

Loc.

dNSA

F-pK

/ dN

SAF+

pKPr

evio

us

Loca

tion

Ref

eren

ces

for P

rev.

Lo

c. (P

MID

)

Pred

icte

d M

W

(kD

a)

Obs

erve

d M

.W.

(kD

a)

Para

logo

us

Fam

ily

(Cas

jens

20

00)

Num

ber

of

Para

logs

Com

men

tsIm

mun

oge

nici

tyTn

m

utan

t

Lipo

P1.0

(http

://w

ww

.cbs

.dtu

.dk/

serv

ices

/Lip

oP/)

TMH

MM

2.0

(http

://w

ww

.cbs

.dtu

.dk/

serv

ices

/TM

HM

M-2

.0/)

Sign

alP4

.1 g

ram

- pre

dict

ions

(h

ttp://

ww

w.c

bs.d

tu.d

k/se

rvic

es/S

igna

lP/)

in v

ivo

Diff

eren

tial E

xpre

ssio

n

Tabl

e 1

CYT

-0.20091

0.08

00o

0.107

100.107

400.125

350.09

0.099N

0.57

SignalP-noTM

CYT

-0.20091

20.09

4.89

0o

0.121

210.111

110.171

10.119

0.115N

0.57

SignalP-noTM

CYT

-0.20091

7.65

7.65

0o

0.202

390.275

390.577

290.35

0.31

N0.57

SignalP-noTM

CYT

-0.20091

0.39

00o

0.111

320.116

320.158

290.099

0.108N

0.57

SignalP-noTM

CYT

-0.20091

0.25

0.08

0i

0.117

400.135

110.207

80.183

0.158N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

310.101

650.11

50.094

0.098N

0.57

SignalP-noTM

CYT

-0.20091

3.03

2.72

0o

0.122

580.135

110.236

10.183

0.158N

0.57

SignalP-noTM

CYT

-0.20091

0.77

0.61

0o

0.111

530.103

590.116

520.088

0.096N

0.57

SignalP-noTM

CYT

-0.20091

4.13

4.13

0i

0.113

360.138

200.282

150.17

0.153N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.02

0o

0.111

370.144

110.301

10.198

0.17

N0.57

SignalP-noTM

CYT

-0.20091

1.3

0.25

0o

0.125

360.136

360.263

320.159

0.146N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.107

450.104

310.121

560.101

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.94

00o

0.109

540.112

160.156

310.124

0.118N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.105

330.157

110.303

30.25

0.201N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0i

0.105

430.108

430.139

390.095

0.102N

0.57

SignalP-noTM

CYT

-0.20091

0.34

0.06

0o

0.143

320.148

410.342

390.146

0.147N

0.57

SignalP-noTM

CYT

-0.20091

6.73

0.34

0o

0.12

260.137

350.251

100.153

0.145N

0.57

SignalP-noTM

CYT

-0.20091

0.07

0.06

0o

0.107

260.129

110.194

80.173

0.15

N0.57

SignalP-noTM

CYT

-0.20091

0.03

0.01

0o

0.122

380.117

120.162

10.134

0.125N

0.57

SignalP-noTM

CYT

-0.20091

00

0i

0.119

380.106

380.106

640.093

0.1N

0.57

SignalP-noTM

CYT

-0.20091

13.23

00o

0.112

420.126

110.249

10.154

0.139N

0.57

SignalP-noTM

CYT

-0.20091

2.89

00o

0.104

510.111

110.159

30.124

0.117N

0.57

SignalP-noTM

CYT

-0.20091

0.3

0.15

0o

0.107

660.103

350.141

20.102

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.11

00o

0.162

190.158

190.198

110.134

0.146N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.128

470.107

110.145

20.112

0.11

N0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.101

300.125

110.2

20.153

0.138N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.137

380.144

110.349

10.185

0.163N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.104

520.104

110.125

10.11

0.107N

0.57

SignalP-noTM

CYT

-0.20091

0.03

0.03

0o

0.105

290.108

600.149

10.098

0.103N

0.57

SignalP-noTM

CYT

-0.20091

16.35

1.55

0o

0.153

470.205

470.43

350.223

0.214N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

660.107

240.129

170.105

0.106N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.105

550.107

440.121

150.099

0.103N

0.57

SignalP-noTM

CYT

-0.20091

0.17

0.17

0o

0.114

320.132

200.185

10.162

0.146N

0.57

SignalP-noTM

CYT

-0.20091

0.25

00o

0.106

460.114

460.146

390.11

0.112N

0.57

SignalP-noTM

CYT

-0.20091

0.06

00o

0.122

290.108

290.155

10.09

0.099N

0.57

SignalP-noTM

CYT

-0.20091

0.1

00o

0.101

320.101

320.121

30.097

0.099N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00i

0.387

220.206

220.136

200.104

0.158N

0.57

SignalP-noTM

CYT

-0.20091

0.19

0.19

0o

0.108

360.121

280.193

230.14

0.13

N0.57

SignalP-noTM

CYT

-0.20091

0.5

0.33

0o

0.104

210.151

210.257

90.215

0.181N

0.57

SignalP-noTM

CYT

-0.20091

0.1

0.01

0o

0.122

500.131

500.27

490.118

0.124N

0.57

SignalP-noTM

CYT

-0.20091

6.73

2.55

0o

0.185

230.33

230.668

150.533

0.425N

0.57

SignalP-noTM

CYT

-0.20091

0.04

0.04

0o

0.105

290.137

110.247

30.194

0.164N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.112

240.104

240.131

100.1

0.102N

0.57

SignalP-noTM

CYT

-0.20091

3.35

00o

0.103

550.126

110.21

40.155

0.14

N0.57

SignalP-noTM

CYT

-0.20091

0.34

0.34

0o

0.133

210.186

190.325

170.253

0.211N

0.51

SignalP-TM

CYT

-0.20091

0.1

00o

0.102

210.11

210.149

240.117

0.113N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.102

180.123

110.228

20.156

0.138N

0.57

SignalP-noTM

CYT

-0.20091

2.82

2.81

0o

0.116

440.182

110.397

30.325

0.249N

0.57

SignalP-noTM

CYT

-0.20091

5.84

4.86

0o

0.113

330.132

210.199

280.151

0.141N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00i

0.101

440.134

110.215

30.187

0.159N

0.57

SignalP-noTM

CYT

-0.20091

0.47

0.38

0o

0.127

470.176

210.392

120.279

0.225N

0.57

SignalP-noTM

CYT

-0.20091

3.44

3.39

0o

0.118

330.159

170.309

130.248

0.201N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.108

300.104

110.147

30.111

0.108N

0.57

SignalP-noTM

CYT

-0.20091

3.89

3.89

0o

0.111

330.156

210.274

130.221

0.186N

0.57

SignalP-noTM

CYT

-0.20091

1.22

1.2

0o

0.146

290.178

290.311

130.22

0.198N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.1

430.109

450.135

130.096

0.103N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.121

450.107

220.134

140.094

0.101N

0.57

SignalP-noTM

CYT

-0.20091

0.02

0.02

0o

0.106

260.118

260.23

180.129

0.123N

0.57

SignalP-noTM

CYT

-0.20091

0.01

0.01

0o

0.252

210.26

210.483

190.258

0.259N

0.57

SignalP-noTM

CYT

-0.20091

0.96

0.78

0o

0.134

470.167

240.343

230.219

0.191N

0.57

SignalP-noTM

CYT

-0.20091

0.03

00o

0.101

540.104

690.112

610.092

0.098N

0.57

SignalP-noTM

CYT

-0.20091

0.01

00o

0.111

660.105

660.127

400.093

0.099N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.121

450.107

220.134

140.094

0.101N

0.57

SignalP-noTM

CYT

-0.20091

1.474

0.02

0.02

0o

0.277

210.3

210.575

190.324

0.311N

0.57

SignalP-noTM

CYT

-0.20091

1.14

1.14

0o

0.125

400.18

240.331

120.241

0.208N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

550.114

110.212

10.123

0.118N

0.57

SignalP-noTM

CYT

-0.20091

1.48

1.47

0o

0.106

510.12

140.169

80.144

0.131N

0.57

SignalP-noTM

CYT

-0.20091

0.02

00o

0.109

660.105

660.13

400.094

0.1N

0.57

SignalP-noTM

CYT

-0.20091

1.52

1.5

0o

0.132

330.178

240.346

230.242

0.208N

0.57

SignalP-noTM

CYT

-0.20091

00

0o

0.1

320.101

320.111

220.09

0.096N

0.57

SignalP-noTM

CYT

-0.20091

6.8

6.69

0o

0.101

330.107

110.142

10.115

0.11

N0.51

SignalP-TM

CYT

-0.20091

00

0o

0.1

210.115

350.146

290.113

0.114N

0.57

SignalP-noTM

CYT

-0.20091

4.43

1.66

0o

0.134

400.189

240.384

230.263

0.224N

0.57

SignalP-noTM

CYT

-0.20091

8.38

8.36

0o

0.106

320.182

130.415

90.332

0.252N

0.57

SignalP-noTM

CYT

-0.20091

0.15

00o

0.102

700.114

110.21

10.121

0.117N

0.57

SignalP-noTM

12

140

References 1. Benach JL, Garcia-Monco JC. 2010. The Worldwide Saga of Lyme Borreliosis. In

Radolf JD, Samuels DS (ed), Borrelia: Molecular Biology, Host Interaction and Pathogenesis. Caister Academic Press.

2. Wormser GP, Wormser V. 2016. Did Garin and Bujadoux Actually Report a Case of Lyme Radiculoneuritis? Open Forum Infect Dis 3:ofw085.

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