biogenesis of outer membrane vesicles in serratia ... · biogenesis of outer membrane vesicles in...

Biogenesis of Outer Membrane Vesicles in Serratia marcescens IsThermoregulated and Can Be Induced by Activation of the RcsPhosphorelay System

Kenneth J. McMahon,a Maria E. Castelli,b Eleonora García Vescovi,b and Mario F. Feldmana

Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Alberta, Canada,a and Instituto de Biología Molecular y Celular de Rosario (IBR),Consejo Nacional de Investigaciones Científicas y Técnicas, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional deRosario, Suipacha, Rosario, Argentinab

Outer membrane vesicles (OMVs) have been identified in a wide range of bacteria, yet little is known of their biogenesis. It hasbeen proposed that OMVs can act as long-range toxin delivery vectors and as a novel stress response. We have found that theformation of OMVs in the Gram-negative opportunistic pathogen Serratia marcescens is thermoregulated, with a significantamount of OMVs produced at 22 or 30°C and negligible quantities formed at 37°C under laboratory conditions. Inactivation ofthe synthesis of the enterobacterial common antigen (ECA) resulted in a hypervesiculation phenotype, supporting the hypothe-sis that OMVs are produced in response to stress. We demonstrate that the phenotype can be reversed to wild-type (WT) levelsupon the loss of the Rcs phosphorelay response regulator RcsB, but not RcsA, suggesting a role for the Rcs phosphorelay in theproduction of OMVs. MS fingerprinting of the OMVs provided evidence of cargo selection within wild-type cells, suggesting apossible role for Serratia OMVs in toxin delivery. In addition, OMV-associated cargo proved toxic upon injection into the hae-mocoel of Galleria mellonella larvae. These experiments demonstrate that OMVs are the result of a regulated process in Serratiaand suggest that OMVs could play a role in virulence.

Gram-negative bacteria, both pathogenic and nonpathogenic,release lipid bilayer vesicles from the outer membrane (OM)

(3, 34). These outer membrane vesicles (OMVs), like the outermembrane from which they are generated, are composed of lipo-polysaccharide (LPS), phospholipids, and outer membrane pro-teins, in addition to periplasmic components (17, 22, 24, 26, 30).Several studies suggest that vesiculation serves as an envelopestress response. Impairment of the �E or the Cpx stress controlmechanisms in Escherichia coli was shown to significantly increasevesiculation, and hypervesiculation was associated with improvedcell survival under stress-inducing conditions (46). OMVs alsohave the capacity to transport damaging agents such as antibiotics(52) or misfolded proteins (46) away from the bacterial cell. Stresscaused by the host environment or by antibiotic treatment canalso result in increased vesiculation in Pseudomonas aeruginosaand Shigella dysenteriae, respectively (10, 23). Within the contextof the host environment, OMVs have been implicated as vectorsfor long-distance toxin delivery (45). This occurs either with thebiogenesis of OMVs acting as a novel secretion mechanism or byshed vesicles associating with secreted proteins and acting as me-diators for these exoproteins (12). Unlike the traditional, highlycharacterized type I to VI secretion systems, OMVs have the ca-pacity to transport a mixture of soluble and insoluble material,which may be advantageous to the bacteria by protecting theircargo from proteases or by allowing for the assembly of multiva-lent complexes. Once released from bacteria, the vesicles are freeto interact with the host, with considerable evidence that OMVscan be internalized into host cells (9, 16, 31) and have a role inmodulating the host immune response (4, 49, 54, 61).

The mechanism for vesicle biogenesis remains elusive. In Por-phyromonas gingivalis and in other species, OMVs are enriched invirulence factors (21, 22). It appears that these proteins are selec-tively packed during OMV biogenesis, in a process that also causes

the enrichment of certain surface glycans and deacylated lipid A inthe vesicles. Studies on P. aeruginosa OMVs have found that thequorum signaling molecule, PQS, is incorporated into the OMand can induce curvature (43, 44). This supports the theory thatthe breakdown of linkages between the peptidoglycan and the OMis the trigger for vesicle formation (35). Alternatively, a nanopod-like structure has also been implicated in vesicle formation in Delf-tia spp., but evidence for this process in other bacteria has yet to befound (55).

OMVs are also produced by Serratia marcescens, a Gram-neg-ative bacillus commonly found in soil, water, air, plants, and ani-mals. S. marcescens is an important nosocomial pathogen and hasbeen implicated as a causative agent in a wide variety of infections,including respiratory and urinary tract infections, septicemia,meningitis, and pneumonia, with significantly increased morbid-ity associated with newborns (2). Serratia has become the subjectof increased attention due to a rise in resistance to several antibi-otics (41, 59) through a series of well-characterized multidrugefflux pumps (1) and porins (51), rendering the pathogen difficultto control and eradicate. Despite its prevalence, the mechanismsof Serratia pathogenesis remain poorly understood. Recently, atype VI secretion system has been described which displays animportant role in bacterial killing (47), and the mechanism forsurvival within human epithelial cells postinternalization was de-

Received 9 January 2012 Accepted 30 March 2012

Published ahead of print 6 April 2012

Address correspondence to Mario F. Feldman, [email protected].

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00016-12

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scribed (14). The outer membrane vesicles of S. marcescens havebeen reported to be among the largest in Enterobacteriaceae (37),but little is known of their composition, their biogenesis, or apossible role in pathogenesis.

Enterobacterial common antigen (ECA) is a glycolipid foundin the outer leaflet of the outer membrane in Gram-negative en-teric bacteria. It has been shown that alterations to this pathway inS. marcescens induce the cell envelope stress-responsive Rcs phos-phorelay system (8). The Rcs phosphorelay is a complex signalingpathway known to be activated upon alterations to lipopolysac-charide or membrane-derived oligosaccharides, with the outermembrane lipoprotein RcsF identified as playing an importantrole channeling selected input signals to the other components ofthe phosphorelay (11, 36, 40). The pathway has a number of com-ponents which require the transfer of a phosphate from the histi-dine kinase domain of RcsC to the phosphotransfer protein RcsD.RcsD can, in turn, transfer the phosphate group to the responseregulator RcsB, which can homodimerize or form complexes withRcsA. The cascade is known to regulate up to 5% of the E. coligenome (15, 19), and in S. marcescens, it has been shown to controlthe expression of several genes, including flagellar biogenesis reg-ulatory components (8). In this work, we have found that OMVbiogenesis is thermoregulated and obtained evidence that, undercertain conditions, RcsB controls an unknown mechanism whichregulates vesiculation in S. marcescens.

MATERIALS AND METHODSBacterial strains and plasmids. Serratia marcescens RM66262 is a non-pigmented clinical isolate from a patient with a urinary tract infection (7).Strains were grown in Luria broth (LB) unless otherwise stated. Gentami-cin and kanamycin were used at 10 �g/ml and 50 �g/ml, respectively. Adescription of the strains used in this study can be found in Table S1 in thesupplemental material.

OMV preparation. Cells corresponding to 55 OD600 units (OD600 isoptical density at 600 nm) of overnight cultures of S. marcescens wild-type(WT) and mutant strains were removed from suspension by centrifuga-tion at 6,000 � g. The supernatants were filtered through a 0.22-�m-pore-size polyvinylidene difluoride (PVDF) membrane (Millex GV; Millipore)to remove residual cells. OMVs were recovered from the resulting filtratesby ultracentrifugation at 100,000 � g for 3 h at 4°C (Optima L-90K ultra-centrifuge; Beckman Coulter) and resuspended in 500 �l of phosphate-buffered saline (PBS) (24).

Kdo quantification. 3-Deoxy-D-manno-octulosonic acid (Kdo) wasevaluated by the periodic acid-thiobarbituric acid method described byHancock and Poxton (20). Briefly, after purification by ultracentrifuga-tion, the vesicles are hydrolyzed by sulfuric acid for 8 min. Periodic acid(0.1 M) is added and the sample left at room temperature for 10 minbefore the addition of sodium arsenite resuspended in HCl. The samplesare vortexed vigorously before the addition of thiobarbituric acid. Afterboiling for 10 min, butanol is added, followed by centrifugation for 4 min.The upper aqueous phase is read at 509 nm (OD), and the reading issubtracted from the values obtained at 552 nm and compared to a stan-dard curve for quantification. Purified Kdo is obtained from Sigma-Al-drich.

Galleria mellonella killing assay. Surface-sterilized G. mellonellaworms were injected into the hind proleg with 5 �l of OMVs or LPS. LPSand OMVs were normalized to 2 ng/�l. Death was then determined after12 h by responsiveness to touch. Worms were examined in triplicates of 30with separate larval preparations. Non-OMV-attributable deaths were de-termined by the injection of worms with sterile PBS.

OM isolation. The cells of overnight cultures were harvested by cen-trifugation at 10,000 � g for 10 min at 4°C. The pellets were gently resus-pended in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 50 mM MgCl2

containing complete EDTA-free protease inhibitor mixture (Roche Ap-plied Science) and then lysed by sonication. The membranes were col-lected by ultracentrifugation at 100,000 � g for 1 h at 4°C. The OMs wereisolated by differential extraction with the same buffer and 1.5% (vol/vol)Triton X-100 and incubated at 20°C for 1 h. The OM fractions wererecovered by centrifugation at 100,000 � g for 1 h at 4°C.

Transmission electron microscopy (TEM). Portions (3 �l) of theOMV preparations were adsorbed onto carbon-coated copper grids (3min). Liquid excess was discarded, and the samples were negativelystained with 2% (wt/vol) uranyl acetate for 3 min and evaluated in aMorgagni (FEI) transmission electron microscope.

Proteomic analysis. Two methods were used to screen for proteincontent of OMVs. The first involved resolving OMV samples by SDS-PAGE followed by the in-gel digestion of excised bands of interest usingsequencing-grade modified trypsin (Promega) (56). Peptide fragmentswere eluted from the gel piece, desalted using ZipTipC18 (Millipore) ac-cording to the supplier’s protocol, and dissolved in 0.1% formic acid. Thesecond method involved the removal of attached lipids from lyophilizedOMV pellets using trifluoroethanol. In-solution trypsin digestion wasthen carried out as before. A hybrid quadrupole orthogonal accelerationtime-of-flight mass spectrometer, Q-TOF Premier (Waters), equippedwith a nanoACQUITY ultraperformance liquid chromatography system(Waters) was used for tandem mass spectrometry (MS-MS) analyses ofthe peptides, and the resulting mass spectrums were used for the identifi-cation of the proteins by the Mascot search engine using the preliminarygene sequence of S. marcescens DB11 on the SEED server.

LPS analysis. LPSs derived from the OMVs of all strains were preparedas described previously (42). The LPS was run on 12% SDS-PAGE andvisualized by the silver staining method described by Tsai and Frasch (60).

RESULTSOMV production in S. marcescens is thermoregulated. Dryweight measurements are often used to compare relative vesicula-tion. The amount of extracellular material, including vesicles, re-covered from the cell-free supernatant of cells grown at 37°C wasapproximately half that which was recovered from cells grown atlower temperatures (Fig. 1, inset). This technique is not accuratefor OMV quantification, as it also measures nonvesicle content,like flagella, which often copurify with OMVs. We consequentlymeasured the 3-deoxy-D-manno-octulosonic acid (Kdo) presentin our OMV preparations. Kdo is an exclusive component of LPSand therefore better reflects the OMV content present in a sample.When OMV production was estimated measuring Kdo, vesicula-

FIG 1 Vesiculation is thermoregulated in S. marcescens. Amounts of OMVwere estimated by measuring Kdo content or dry weight (inset). Both methodsshow that production of OMV is higher at 22 or 30°C than at 37°C and thatlittle difference is noted between 22°C and 30°C in terms of OMVs recovered.Average Kdo values were calculated from five separate experiments, and theerror bars indicate standard deviations.

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tion was diminished about 5-fold at 37°C compared to that at 22or 30°C (Fig. 1). This indicates that in S. marcescens, OMV pro-duction is thermoregulated, being high at low temperatures andminimal at high temperatures. As an additional control, we deter-mined that Kdo levels were not variable on the surface of wholecells at each of the temperatures (see Fig. S1 in the supplementalmaterial).

Disruption of the ECA synthesis pathway increases vesicula-tion at all temperatures. In searching for genes that affect vesicu-lation in S. marcescens, elements of the ECA synthesis pathwaywere examined. WecG converts GlcNAc–PP-Und (lipid I) toManNAc-GlcNAc–PP-Und (lipid II), with mutagenesis of thewecG gene resulting in the accumulation of lipid I at the innermembrane. Previous work has shown that bacteria carrying mu-tations to the ECA synthesis cluster express low levels of flagellin,lack phospholipase activity, and present reduced swimming andswarming compared to wild-type bacteria (7). We found that inthe wecG mutant, vesiculation was induced at all temperatures(Fig. 2). The increase was approximately 2- to 3-fold at 22°C and30°C and about 8-fold at 37°C. Interestingly, the wecG mutantproduced at 37°C more vesicles than the wild-type strain at 22°Cor 30°C. The complementation of the wecG mutant restored OMVproduction to levels comparable with those of the wild type. In-vestigation of the purified OMVs by TEM revealed that OMVsproduced by the three strains at 37°C were similar in shape andaveraged 150 nm in diameter (Fig. 2, inset). Though we acknowl-edge that larger OMVs may be absent from our preparations dueto our harvesting method, the uniform size and appearance of theOMVs seen by TEM suggest that the majority of the OMVs areapproximately 150 nm in size. Consistent with the data obtainedvia Kdo determination, the amounts of vesicles visualized in thewecG strain were higher than for the wild-type or the comple-mented strains.

ECA synthesis disruption at different stages increases vesic-ulation. We analyzed if the hypervesiculation phenotype detected

in the wecG mutant was also observable by disrupting other genesinvolved in ECA synthesis, as occurs for the other phenotypespreviously described for ECA mutants (7, 8). wecA and wecDmutations of the ECA synthesis pathway result in the accumula-tion of undecaprenyl monophosphate (Und-P) and lipid II inter-mediates, respectively. Both mutations resulted in increased vesic-ulation at 30°C and 37°C, indicating that temperature-inducedvesiculation and �ECA-induced vesiculation may be differentprocesses. The degree of increase varied, being more dramatic inthe wecD mutant (Fig. 3). The approximately 20-fold increase ofvesiculation apparent by comparison of the wild type and wecDmutant at 37°C and the 6- to 8-fold increase in the case of the wecAand wecG mutants are clear indications of the effect of altering theECA synthesis pathway on vesiculation. TEM of the OMVs wascarried out, with intact OMVs from the wecD and wecA strainsappearing similar to the ones produced by the wild-type strain(Fig. 3, insets A and B). To rule out that cell lysis accounted for theapparent hypervesiculation phenotype observed, MS fingerprint-ing of the proteins present in our OMV preparations was carriedout. The vast majority of the proteins found were outer membraneand periplasmic proteins (Tables 1 and 2). The cytoplasmic pro-teins alcohol dehydrogenase and EF-Tu were the only predictedcytoplasmic proteins present within both OM and OMV prepara-tions. This finding, in addition to the absence of inner membraneproteins, argues against lysis. The LPSs of the OMVs of all strainswere compared to ensure that the surface carbohydrate waspresent, a characteristic of all OMVs. As a final control to ensurethat broken cells were not a contaminant of the OMV prepara-tions, Western blot analysis of trichloroacetic acid (TCA)-precip-itated OMV-free supernatants was carried out using �-RNA poly-merase. The presence of cytoplasmic RNA polymerase in thesupernatant would be indicative of cell lysis. No significant levelsof RNA polymerase were detected in the supernatants, ruling outa contribution of cell lysis to the amounts of Kdo measured (datanot shown).

FIG 2 Interruption of the ECA synthesis pathway results in hypervesiculation. The amount of Kdo released from stationary-phase cells increased upon mutationof wecG. Reintroduction of wecG restored wild-type levels of vesiculation. Average Kdo values were calculated from five separate experiments, and the error barsindicate standard deviations. Also shown is TEM analysis of vesicles (indicated by arrows) in WT (inset A), wecG (inset B), and wecG (inset C) samples at 37°C.The scale bar present at the bottom left of each image corresponds to 200 nm.

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RcsB and not RcsA is necessary for hypervesiculation in theECA-deficient background. As mentioned earlier, mutating theECA synthesis pathway has been previously shown to activatethe Rcs phosphorelay (8). We analyzed if the hypervesiculationphenotype observed was dependent on the Rcs phosphorelaypathway by comparing the amounts of OMV produced by thewecD mutant and double wecD-rcsB and wecD-rcsA mutants. At37°C, the hypervesiculation phenotype resulting from the intro-duction of a mutation into the ECA synthesis pathway was sup-pressed through the introduction of a secondary mutation in rcsB(Fig. 4A), with over 20 times the quantity of OMVs recovered inthe wecD strain compared to the rcsB-wecD double mutant at thistemperature. Conversely, vesiculation was not restored to WT lev-els in a wecD-rcsA double mutant. This indicated that within theRcs phosphorelay, rcsB is the response regulator responsible forhypervesiculation at 37°C. At 30°C, however, the phenotype wasnot restored by the introduction of a secondary mutation in rcsB,suggesting that a factor unrelated to rcsB is involved in this process(Fig. 4A). To discount the effect of temperature on OMV produc-tion caused by disruption of ECA synthesis, we estimated vesicu-lation at 30°C and 37°C in terms of fold increase or decrease rela-tive to their respective WT levels (Fig. 4B and C). This analysisrevealed a 20-fold increase in vesiculation at 37°C, compared justa 3-fold increase at 30°C in the wecD mutant strain relative to WTlevels (Fig. 4B and C). It is known that temperature has a markedeffect on the lifestyle of the organism, with several genes alreadyidentified as being expressed differently at 30°C and 37°C (38, 62).At 30°C and 37°C, rcsB and rcsA single mutants display amounts ofvesiculation comparable to that of the WT strain, suggesting thatthe loss of this Rcs response regulator does not result in stress-induced vesiculation.

Proteomic analysis of S. marcescens OMVs. It has been re-ported that protein cargo selection occurs in P. gingivalis upon theformation of OMV and that this process requires the integrity ofthe LPS (21). The enrichment or exclusion of outer membraneproteins from OMVs has also been reported for other bacteria, buta mechanism has not been identified (22, 28, 63). We tested ifcargo selection in S. marcescens OMV takes place by comparing

the protein compositions of OMV and OM at 30°C. This condi-tion was chosen because at 30°C, vesiculation is activated in WTcells. The OMs and OMVs were harvested from overnight culturesand the protein profile analyzed by SDS-PAGE. The major bandspresent in Fig. 5 were identified by liquid chromatography-MS(LC-MS). MS analysis of the entire proteomic content of eachfraction also showed that each fraction contained a different sub-set of proteins. For example, OmpA, OmpW, and OmpX seemedto be enriched in the OMV fraction (Fig. 5). The extracellularprotease serralysin also appeared to be enriched in the OMV frac-tion. However, because this protein is secreted via a type I secre-tion system before its association to OMVs, its presence in theOMV fraction does not reflect a preferential sorting of this proteininto the OMVs (29). To perform a more comprehensive compar-ison between the protein contents of OMVs and OMs, samplescontaining purified vesicles and OMs were subjected to trypsindigestion and fingerprinting by MS (Fig. 6 and Tables 1 and 2).This analysis can detect proteins present in minimal amounts butonly indicates the presence or absence of proteins, without differ-entiating levels of proteins in the samples. Several outer mem-brane proteins, like TolC, LptD, maltoporin, and YaeT, were de-tected in the OM and not in the OMV fractions. It is tempting tospeculate that these proteins are excluded from the vesicles duringOMV biogenesis. A number of proteins were detected in OMVsamples only. These are the outer membrane protein MipA andperiplasmic alanine amidase and extracellular proteins like lipaseA, phospholipase, and serralysin. Due to their localization to theperiplasm, none of these proteins are expected to be found in OMfractions (29). Lipase A and phospholipase, like serralysin, likelyassociate with OMVs after their secretion into the media. Takentogether, these data suggest that some proteins are enriched inOMVs and others are excluded from OMVs. These data supportthe hypothesis of cargo selection in OMVs that has been made forother organisms (21, 22).

OMVs from WT cells are toxic to Galleria mellonella inde-pendently of LPS. It has been proposed that OMVs may play a roleas vectors for the long-distance delivery of virulence determi-nants. To determine if the OMVs of S. marcescens carry active

FIG 3 Interruption of the ECA synthesis pathway at various stages induced OMV formation. Kdo recovered from cell-free supernatants shows increasedamounts of vesicles in wecA, wecD, and wecG strains. wecA (inset A) and wecD (inset B) OMVs recovered from 30°C cultures of wecA and wecD strains appearsimilar to OMVs isolated at 37°C from WT and wecG strains (Fig. 2). The scale bar present at the bottom left of each image corresponds to 200 nm. Average Kdovalues were calculated from five separate experiments, and the error bars indicate standard deviations.

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toxic components, we investigated their toxicity to larvae of thegreater wax moth, Galleria mellonella. The immune response ofthe larvae has been well characterized, with haemocoel coagula-tion, cellular phagocytosis, and phenol oxidase-based melaniza-tion employed to defend from infection. Toll-like receptors andpeptidoglycan receptors are also present for the recognition ofinvasive microbes (53). We investigated the effect of the injectionof a defined amount of OMVs into the haemocoel of the larvae.Figure 7 shows that OMVs collected at both 30°C and 37°C, in anamount equivalent to 10 ng of Kdo and 500 to 750 ng of proteins,resulted in 100% larval death within 24 h. The toxicity was inde-pendent of LPS, as the same amount of LPS did not cause a similareffect, with more than 75% survival after 24 h. There was no sig-nificant difference between the toxicities of WT OMVs derivedfrom cells grown at 30°C and at 37°C.

DISCUSSION

For several decades, OMVs were considered to be microscopyartifacts and/or cell debris. However, in the last years several re-ports have shown that OMV biogenesis is a fine-tuned process ina number of Gram-negative bacteria. In this work we showed thatS. marcescens produces large amounts of OMVs at 22 or 30°C,while negligible quantities of OMVs are produced at 37°C. To ourknowledge, the identification of thermoregulation control inOMV biogenesis in Gram-negative bacteria is unprecedented, andthis finding adds to the evidence that vesiculation is an active, anda highly regulated, process.

We observed an increase in vesiculation by mutating genesinvolved in the ECA synthesis pathway. Despite the presence ofECA on the surface of all Gram-negative enteric bacteria, its role is

TABLE 1 Proteins detected in the OM and OMVs of wild-type S. marcescensa

Fraction Systematic gene name Annotation MW Score

OMV 4161L Lipase 64.9 1,3876253L Flagellin 34.6 1,10312577R Serralysin A 54 8792860L OmpF 41.7 7192408R Surface layer protein A 101.2 6392927L OmpA 39.2 4397297L Alanine amidase 28.4 3516014L Porin 39.6 2274103L Lipoprotein precursor 8.3 1477646L OmpC 41.4 1615558L OmpW 23.8 1396463R Hcp effector 16.8 1385731L MipA protein 28.5 1271470R Peptidoglycan-associated lipoprotein 19.4 12312753R EF-Tu 43.2 1102143R OmpX 18.4 1108985R Phospholipase 35.6 1035653R Alcohol dehydrogenase 96.2 86

OM 2927L OmpA 39.2 1,4942860L OmpF 41.7 1,03413014R GroEL 56.6 8956253L Flagellin 36.6 8937646L OmpC 41.4 8326014L Porin 39.6 4985558L OmpW 23.8 4431470R Peptidoglycan-associated lipoprotein 19.4 4346011L LysM 34.5 4265653R Alcohol dehydrogenase 96.2 3294176L OM lipoprotein 16.6 2974103L Lipoprotein precursor 8.3 29210953L Ribosomal 30S 23.6 2209153L Omp factor YaeT 89.6 19310162R TolC 54.5 190109L LPS assembly protein LptD 89.3 1876322L Flagellar hook 42.6 18712753R EF-Tu 43.2 1652143R OmpX 18.4 1452408R Surface layer protein A 101.2 14112670R Ribosomal 50S 24.6 12410954L Ribosomal 30S 17.6 12211000L Ribosomal S7 13.9 1179916L Dihydrolipoamide dehydrogenase 43.9 107

a Proteins detected by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The score contributed to protein hits can also be used to estimate theirprevalence within the sample. In bold, extracellular or hypothesized extracellular proteins are identified. MW, molecular weight.




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TABLE 2 Proteins detected in the OM and OMVs of WecD mutant strains of S. marcescensa

Fraction Systematic gene name Annotation MW Score

OMV 6253L Flagellin 34.6 9205558L OmpW 23.8 69712753R Ef-Tu 43.2 6244161L Lipase 64.9 62210801L Maltoporin 48.2 5967646L OmpC 41.4 5466014L Porin 39.6 5338985R Phospholipase 35.6 52312577R Serralysin A 54.0 5195653R Alcohol dehydrogenase 96.2 3172860L OmpF 41.7 3502408R SLA 101.2 3267297L Alanine amidase 28.4 30512364R Chitinase 61.0 2735731L MipA protein 28.5 2731470R Peptidoglycan-associated lipoprotein 19.4 2599153L Omp factor YaeT 89.6 2602927L OmpA 39.2 25013014R GroEL 56.6 2493763R Tsx 33.4 2341467R TolB 46.0 2274103L Lipoprotein precursor 8.3 19810350R Hypothetical protein 19.9 191409R Lipoprotein NlpD 35.0 185

OM 4161L Lipase 64.9 1,0026253L Flagellin 34.6 92912577R Serralysin A 54 8792927L OmpA 39.2 7981467R TolB 46.0 62910814R Putative membrane protein 56.9 5977646L OmpC 41.4 5372408R SLA 101.2 5308612L OM autotransporter 102.3 4767297L Alanine amidase 28.4 4741283L Glutamate transporter 33.1 4182860L OmpF 41.7 3549153L OM assembly protein 89.6 349960L Putative lipoprotein 20.7 3395558L OmpW 23.8 33410801L Maltoporin 48.2 2981013R Putative lipoprotein 16.9 2912143R OmpX 18.4 2893535L LolB 25.0 2814561R Serralysin B 57.7 2704103L Lipoprotein precursor 8.3 26811524L SSph2 107.5 2613763R Tsx 33.4 2375731L MipA protein 28.5 2319153L Omp factor YaeT 89.6 2206463R Hcp1 16.8 2135643L Periplasmic oligopeptide-binding protein 61.5 2041473R Tol-Pal protein YpgF 28.1 201409R Lipoprotein NlpD 35.0 1963237L YceI 21.3 1901470R Peptidoglycan-associated lipoprotein 19.4 1644176L OM lipoprotein 16.6 1624456R Peptidyl-dipeptidase Dcp 82.4 1566014L Porin 39.6 1516011L LysM 34.5 15110162R TolC 54.5 15012369R Extracellular solute-binding protein 64.5 142

a In bold, extracellular or hypothesized extracellular proteins are identified. Only proteins scoring above 100 are listed. The S. marcescens Db11 genome can be found at http://www.sanger.ac.uk/Projects/S_marcescens/. MW, molecular weight.

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relatively unknown. ECA mutant strains of Salmonella entericawere found to have lower resistance to bile and were attenuated inoral and interperitoneal mouse models of infection, though it re-mains possible that this was a secondary result of the accumula-tion of undecaprenyl monophosphate or lipid II intermediates(18, 50). The mutation of wecA, wecD, and wecG eliminates allforms of ECA, of which there are three forms, cyclic periplasmic,LPS anchored, and linked to phosphatidylglycerol (13, 25). Figure4 shows that at both 37°C and 30°C, the mutations to wecA, wecD,and wecG, despite resulting in the accumulation of different lipidintermediates, all have upregulated vesiculation levels, suggestingthat it is the loss of one or all forms of ECA rather than the accu-mulation of undecaprenol monophosphate, lipid I, or lipid II in-termediates that causes the hypervesiculation. The mutation ofthese genes results in the activation of rcsB at 37°C (8), but the lossof rcsB in the ECA-deficient background results in low levels ofvesiculation, comparable to those of the wild type, at 37°C (Fig. 5).

A wide variety of other stimuli that affect the integrity of the cellenvelope are also known to induce the Rcs phosphorelay, includ-ing high osmolarity and the overexpression of some membraneproteins (39). We have also noticed an increase in vesiculation inWT cells grown in high-osmolarity media, suggesting that OMVproduction through RcsB is not specific to ECA loss but a moregeneral mechanism for coping with OM stress (data not shown).The deletion of genes involved in LPS synthesis is known to acti-vate the Rcs phosphorelay in other bacteria (50), and recently, itwas shown that in Vibrio fischeri, the overexpression of a sensorkinase which regulates polysaccharide production also contrib-uted to an increase in vesiculation (57). We could consequentlyspeculate that the cell surface carbohydrates not only are impor-tant for cell recognition and interactions but also play an impor-tant role in maintaining the integrity of the cell envelope andtriggering vesiculation as a stress response.

FIG 4 The loss of rcsB within an ECA mutant strain results in the reversion of the hypervesiculation phenotype. A comparison of the net amount of OMVrecovered from the various strains is displayed in panel A. Panels B (37°C) and C (30°C) display the same data in terms of a fold difference in vesiculation,withWT levels set to 1. Note the difference in scale between panels B and C. These results demonstrate that introduction of a mutation into the rcsB gene reverts thehypervesiculation phenotype caused by the wecD mutation at 37°C. Average Kdo values were calculated from five separate experiments, and the error barsindicate standard deviations.

FIG 5 Protein profiles of the OM and OMVs appear different in WT S. marc-escens. Purified OMs and OMVs were analyzed by silver staining and 15%glycine-PAGE. Cell fractions were collected from WT bacteria grown at 30°Cand 37°C.

FIG 6 Schematic of the compartmentalization of OM and OMV proteins. TheVenn diagram indicates the location of proteins isolated from the OM and theOMVs of wild-type cells grown at 30°C. Proteomic content was determined bytryptic digestion of the entire fraction, followed by MS fingerprinting analysis.




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Thermoregulation within the Rcs phosphorelay has been re-ported before, as the unstable effector rcsA is thought to be morereadily degraded at higher temperatures, rendering Rcs-depen-dent expression of cps genes greatly reduced (58). However, wehave shown that rcsA does not contribute to the increase in vesic-ulation in this case (Fig. 5). Little is known about thermoregula-tion in S. marcescens. The production of the surfactant serrawettinis known to occur only at 30°C (38, 62). The ability of S. marcescensto swarm, swim, and express flagellin is also differentially affectedby growth at 30°C and at 37°C (7). The model that we propose isthat at 37°C, significant vesiculation is not detected, but upondisruption of the ECA synthesis pathway, rcsB is activated andrcsB-regulated gene or genes encoding enzymes involved in OMVbiogenesis are induced, resulting in hypervesiculation. At 30°C, anon-rcsB-dependent mechanism moderately induces vesiculationin wild-type cells. The rcsB DNA recognition sequence has beenidentified, and we have found the recognition site upstream of anumber of important LPS-modifying genes, for example, that forthe lipid A acyltransferase PagP (5). In a recent study, it was de-termined that lipid A had a different acetylation profile in OMVsthan in the OM in P. gingivalis (21). It is tempting to speculate thatRcs-controlled genes like the PagP gene would modify lipid A in S.marcescens, contributing to an increase in vesicle production.

As previously observed for the OMVs of Xenorhabdus nemato-philus (32), the OMVs of S. marcescens were toxic to G. mellonella,supporting the theory that bacterial OMVs can act as vectors fordelivery of virulence factors. The extracellular proteases of S.marcescens were shown to be toxic to G. mellonella (27), and theseare likely the same proteases identified within the OMVs (Tables 1and 2). A large number of other virulence-associated factors(lipases, phospholipases, and chitinases) are present in theseOMVs, and therefore, it is unsurprising that they have the abilityto induce larval death; however, the toxicity of S. marcescens OMVpreparations to G. mellonella has not been demonstrated before,and this simple model provides a useful tool in the characteriza-tion of the role of the OMVs in pathogenesis.

As study into OMV biogenesis progresses, it is likely that fur-ther characterization of the complex interactions between theouter membrane and peptidoglycan will be critical to our under-

standing of the mechanistic requirements of the process. Theidentification of an organism which upregulates and downregu-lates vesiculation through the well-characterized Rcs phosphore-lay could be an important step toward our understanding of thedynamic process of vesiculation. Identification of the key compo-nents of the process could allow for the design of novel antibioticsto inhibit this key step in pathogenesis for many enteric bacteria.Alternatively, the process could be exploited for the improvementof the OMV-based vaccines (6, 48), which may impact global vac-cination strategies.

ACKNOWLEDGMENTS

We thank M. Florencia Haurat for critical reading of the manuscript,Jennifer Ng for technical assistance, and Bela Reiz (Department of Chem-istry, University of Alberta) for assistance with mass spectrometry.

This work was supported by grants from the Alberta Glycomics Cen-tre. E.G.V. and M.E.C. are supported by grants from Agencia Nacional dePromoción Científica y Tecnológica (ANPCyT) and from Consejo Nacio-nal de Investigaciones Científicas y Tecnológicas (CONICET), Argentina.E.G.V. and M.E.C. are career investigators of CONICET, Argentina.M.F.F. is an Alberta Heritage Foundation for Medical Research (AHFMR)scholar and a Canadian Institute for Health Research (CIHR) New Inves-tigator.

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