of March 24, 2018.This information is current as

Complement PathwaysDeposition via the Lectin and ClassicalCIP Interacts with C4, Preventing C3b

Secreted Protein−StreptococcusThe Group B

Scilla Buccato, Pietro Speziale and Immaculada MargaritGiampiero Pietrocola, Simonetta Rindi, Roberto Rosini,

ol.1501954http://www.jimmunol.org/content/early/2015/11/25/jimmun

published online 25 November 2015J Immunol 

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The Journal of Immunology

The Group B Streptococcus–Secreted Protein CIP Interactswith C4, Preventing C3b Deposition via the Lectin andClassical Complement Pathways

Giampiero Pietrocola,* Simonetta Rindi,* Roberto Rosini,† Scilla Buccato,†

Pietro Speziale,* and Immaculada Margarit†

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular

polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can

however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this

study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement

activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b

and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant

CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in

a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for

the development of anti-infective agents against this important pathogen. The Journal of Immunology, 2016, 196: 000–000.

Streptococcus agalactiae (group B Streptococcus [GBS])colonizes the lower gastrointestinal and vaginal mucosaeof about one third of women and can cause neonatal

pneumonia, sepsis, and meningitis (1, 2). It is also an importantetiological agent of morbidity in immunocompromised adults andof bovine mastitis (3). Both during colonization and in the in-fection stage, GBS bacteria are faced with the host innate im-mune defense, and one of the first barriers they encounter is thecomplement system. Several complement effector molecules canindeed sense and opsonize Gram-positive bacteria such as GBSand promote their phagocytic killing by neutrophils and macro-phages (4).The process of complement fixation can occur by three acti-

vation routes, the classical pathway (CP), the lectin pathway (LP),and the alternative pathway (AP), differing in their target recog-nition mechanisms and effector molecules. All three proteolyticcascades lead to cleavage of C3 and subsequent formation of theC3a anaphylatoxin and the C3b opsonin. C3a attracts and activatesgranulocytes, whereas C3b attaches covalently to the bacterial

surface, amplifies complement activation, and labels cells forphagocytosis. Activation of the CP is initiated after C1q moleculesare deposited on the bacterial surface via direct recognition, Igbinding, or pentraxins bridging and interact with C1r and C1sproteases to form the C1 proteolytic complex. Through the LPpathway, mannan-binding lectin or other lectins bind to microbialsurface polysaccharides resulting in activation of mannan-bindinglectin–associated serine protease (MASP). Both of the CP and LPproteolytic complexes can split surface-bound C4 into C4a plusC4b, and C2 into C2b plus the C2a protease. C4b and C2a directlyinteract to form the C3 convertase C4bC2a that cleaves native C3into C3b. Surface-bound C3b is in turn the precursor of C3bBb,the AP C3 convertase that transforms new C3 molecules into C3band C3a, thus greatly amplifying the number of C3b moleculesopsonizing the bacteria and consequently phagocytic killing.Three important host regulators controlling complement ho-

meostasis are C3b-cleaving factor I, factor H, which acts as acofactor of factor I and can also compete with factor B to displaceBb from the AP C3bBb convertase, and C4b-binding protein thatinterferes with the assembly of the CP/LP C4bC2a convertase.Bacterial pathogens have evolved a series of innate defense

evasion molecules that can block the complement proteolyticcascades or divert them to overcome immune clearance by the host.In the case of GBS, a prominent role in complement evasion isplayed by the thick capsular polysaccharide that surrounds thebacterial cell wall. Almost all GBS strains associated with humandisease are encapsulated, belonging to 1 of 10 capsular typesrecognized by specific Abs: Ia, Ib, and II–IX. The 10 GBS capsularpolysaccharide structures are created by diverse arrangementsof galactose, glucose, N-acetylglucosamine, and sialic acid intounique repeating units that invariably contain sialic acid on theirbranching terminus (5). Type III GBS, frequently found in neo-natal invasive infections, expresses a large amount of capsularpolysaccharide that was shown to inhibit activation of the com-plement AP in adult sera deficient in specific Abs (6), whereas APinhibition could be overcome by anti–type III polysaccharide IgG(7). Furthermore, a mutant strain expressing a capsule devoid ofsialic acid showed a markedly decreased virulence (6, 8). Marques

*Department of Molecular Medicine, Unit of Biochemistry, University of Pavia,27100 Pavia, Italy; and †GSK Vaccines S.r.l., 53100 Siena, Italy

ORCID: 0000-0002-7069-8155 (G.P.).

Received for publication September 2, 2015. Accepted for publication October 30,2015.

Address correspondence and reprint requests to Dr. Immaculada Margarit or Prof.Pietro Speziale, GSK Vaccines S.r.l., Via Fiorentina 1, 53100 Siena, Italy (I.M.) orDepartment of Molecular Medicine, Unit of Biochemistry, University of Pavia, VialeTaramelli 3/b, 27100 Pavia, Italy (P.S.) E-mail addresses: immaculada.x.margarit-y-ros@gsk.com (I.M.) or pspeziale@unipv.it (P.S.)

Abbreviations used in this article: AP, alternative pathway; CIP, complement inter-fering protein; CP, classical pathway; Eap, extracellular adherence protein; Efb,extracellular fibrinogen-binding protein; Fib3, fibrinogen-binding protein 3; GAS,group A Streptococcus; GBS, group B Streptococcus; LP, lectin pathway; MASP,mannan-binding lectin–associated serine protease; PBST, PBS supplemented with0.1% (v/v) Tween 20; Sip, group B streptococcal surface immunogenic protein;SPR, surface plasmon resonance.

This article is distributed under The American Association of Immunologists, Inc.,Reuse Terms and Conditions for Author Choice articles.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501954

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et al. (9) confirmed the critical role of the sialic acid–containingcapsule in preventing C3b deposition, presumably through acqui-sition of factor H from host plasma, resulting in cleavage of C3b toiC3b by factor I and interruption of the AP C3b amplification loop.Some of the GBS cell wall surface–anchored proteins containing

a leucine-proline-any-threonine-glycine motif contribute to inhibi-tion of bacterial phagocytic killing by blocking complement acti-vation. These include the b protein, which interacts with the factorH complement regulator (10) and sialic acid–binding Siglec-5 lectinexpressed on leukocyte surfaces (11), the histidine triad proteinSTH, which also binds factor H promoting complement degradation(12), and BibA, a group B Streptococcus immunogenic bacterialadhesin that specifically binds to human C4b-binding protein (13).Studies in the last decade have revealed that another Gram-

positive pathogen, Staphylococcus aureus, produces soluble proteinsinterfering with the activation of the complement system such asstaphylococcal complement inhibitor (14), extracellular fibrinogen-binding protein (Efb) (15, 16), and extracellular adherence protein(Eap) (17). We hypothesized that S. agalactiae might secrete similaryet unidentified complement regulators assisting the bacteria to es-cape phagocytic killing. With this in mind, we screened the GBSgenome for the presence of potentially secreted proteins that coulddisplay inhibitory activities on one or more complement pathways.A low molecular mass protein interfering with the CP and the LPwas identified, and we could demonstrate its capacity to bind the C4complement factor and prevent the formation of the C4bC2a CP/LPproconvertase and GBS phagocytic killing in the absence of anti-GBS Abs.

Materials and MethodsProteins and human-derived materials

Human serum proteins C3b, C4b, and C2 were obtained from Calbiochem(Merck, Darmstadt, Germany). Factor B was from CompTech (Tyler, TX).BSAwas purchased from Sigma-Aldrich (St. Louis, MO). Blood for serumpreparation and for phagocytosis assays was drawn from healthy adultvolunteers after informed consent and approval of the protocol by theMedical-Ethical Committee of the University of Pavia were obtained. Bloodwas allowed to clot for 15 min at 22˚C, and serum was collected aftercentrifugation for 10 min at 1500 3 g at 4˚C and subsequently stored at280˚C. Complement factor–depleted serum was obtained from CompTech.

Bacterial strains and growth conditions

Escherichia coli BL21(DE3) (Stratagene, La Jolla, CA) was used as hostfor expression of recombinant proteins and was grown in Luria brothcontaining ampicillin (100 mg/ml). GBS clinical isolates COH1 (serotypeIII) (18), 515 (serotype Ia) (19), 2603 (serotype V) (20), 6313 (serotype III)(21), 383728 (NT), ES-NI-010 (serotype III), and SH0248 (NT) (22) wereused in this study. Bacteria were grown at 37˚C in Todd-Hewitt broth(Becton Dickinson, Sparks, MD) or in modified M9 medium (28 mMNa2HPO4, 22 mM KH2PO4 [pH 7.4] containing 8.5 mM NaCl, 18.7 mMNH4Cl, 2 mM MgSO4, 1 mM CaCl2, 0.2% glucose, 0.3% yeast extract,and 1% casamino acids).

Expression of the complement interfering protein in E. coli

DNA encoding complement interfering protein (CIP) was amplified byPCR using chromosomal DNA isolated from GBS COH1 as template. Toamplify DNA encoding CIP, forward (59-GGAATTCCTAGCTAGCAA-GAGTGATGGCATCTC-39) and reverse (59-GGAATTCCCG CTCGAG-TCTAAAACTATCTTTTATTACTTT-39) primers were used. Restrictionenzyme cleavage sites NheI and XhoI were incorporated at the 59 ends ofthe primers to facilitate cloning into the pET21b(+) expression plasmid(Novagen, Podenzano, Italy).

Purification of recombinant proteins

An overnight starter culture of the recombinant E. coli expressing CIP wasdiluted 1:50 in Luria broth and incubated at 37˚C with shaking until theculture reached OD600 of 0.6–0.8. Recombinant protein expression was in-duced by addition of isopropyl 1-thio-b-D-galactopyranoside (0.2 mM) andcontinued for 4 h. Bacteria were harvested by centrifugation at 1700 3 g for

20 min and lysed by passage through a French press. The cell debris wasremoved by centrifugation (20,000 3 g), and the filtered supernatant (0.45-mm membrane) was applied to a 1 ml Ni2+-Sepharose HisTrap HP column(GE Healthcare, Buckinghamshire, U.K.). The protein was eluted with 29column vol 0.00–500 mM imidazole (Sigma-Aldrich) gradient in 0.5 MNaCl, 20 mM sodium phosphate (pH 7.4). Fractions corresponding to therecombinant protein were pooled and dialyzed against PBS. Protein con-centrations were determined with a bicinchoninic acid protein assay kit(Pierce, Rockford, IL). Recombinant fibrinogen-binding protein 3 (Fib3) wasexpressed and purified as previously described (23).

Abs

Goat polyclonal anti-human C4 and rabbit polyclonal anti-human C3 Abswere purchased from Abcam (Cambridge, U.K.). HRP-conjugated sec-ondary Abs were from Dako (Glostrup, Denmark). Polyclonal rabbit anti-CIP Ab and mouse anti–group B streptococcal surface immunogenicprotein (Sip) Ab were obtained by immunizing New Zealand rabbits andCD1 mice with three doses of recombinant protein (20 mg). Protocols wereapproved by the Italian Ministry of Health (authorization 110/2012-B) andby the local Novartis Animal Welfare Body (authorization AWB 201114).

Immunoassays for assessment of complement functionalactivity

Wielisa assays (24) were conducted according to the manufacturer’s in-structions (Wieslab, Malmo, Sweden). Briefly, normal human serum (1–6ml) was preincubated for 30 min at 22˚C in 100 ml assay buffer containing0–200 nM of CIP or Fib3 and added to wells coated with IgM, LPS, ormannan. Complement activation was detected by using either alkalinephosphatase–labeled anti–C5-9 complex or anti-C3 (1:2000)– or anti-C4(1:2000)–specific sera, followed by incubation with HRP-labeled second-ary Abs (1:1000). Heat-inactivated serum was used as a negative control.

The EZComplement CH50 clinical diagnostic assay kit (Diamedix,Miami,FL) was used as recommended by the manufacturer. Briefly, 5 ml humanserum was preincubated for 1 h with increasing doses of CIP (0–350 nM) orFib3 (350 nM) and then incubated at 22˚C for 1 h with Ab-coated sheepRBCs (3 ml). Cells were then centrifuged (800 3 g for 10 min) and theabsorbance of the supernatants was measured at 405 nm to determine thepercentage lysis in each sample. The data are expressed as percentage RBClysis in CIP preincubated compared with nonpreincubated samples.

ELISA to assess binding of C4b to CIP

Microtiter wells were coated with 100 ng CIP and incubated overnight at4˚C in 50 mM carbonate buffer (pH 9.5). The wells were washed threetimes with PBS supplemented with 0.1% (v/v) Tween 20 (PBST), blockedwith 2% BSA in PBST for 1 h at 22˚C, and then probed with serial di-lutions of normal human serum in PBS, followed by incubation with goatanti-C4 Ab (1:2000) and HRP-conjugated rabbit anti-goat IgG (1:1000)and detection of HRP enzymatic activity.

SDS-PAGE and Western blotting

SDS-PAGE was performed on 12.5% polyacrylamide gels stained withCoomassie brilliant blue (Bio-Rad, Hercules, CA). For the Western blotassay, CIP was subjected to SDS-PAGE and then electroblotted onto anitrocellulose membrane (GE Healthcare). The membrane was treated witha solution containing 5% (w/v) dried milk in PBS, washed, and incubatedwith 1% human serum for 1 h at 22˚C. Following additional washings withPBST, the membrane was incubated for 1 h with an anti-C4 goat poly-clonal Ab (1:5000). After several washings, the membrane was incubatedwith HRP-conjugated rabbit anti-goat IgG (1:10000), followed by detec-tion of HRP enzymatic activity.

Surface plasmon resonance analysis of CIP binding to C4b

Surface plasmon resonance (SPR) to estimate the affinity of the interaction be-tween C4b and CIP was conducted using a BIAcore X100 instrument (GE LifeSciences). Purified human C4b was covalently immobilized on dextran matrixCM5 sensor chip surface by using a C4b solution (30 mg/ml in 50 mM sodiumacetate buffer [pH 5]) in a 1:1 dilution with N-hydroxysuccinimide and N-ethyl-N9-(3-dimethylaminopropyl)carbodiimide hydrochloride. The excess of activegroups on the dextran matrix was blocked using 1M ethanolamine (pH 8.5). Onanother flow cell, the dextran matrix was treated as described above but withoutany ligand to provide an uncoated reference flow cell. The running buffer usedwas PBS containing 0.005% (v/v) Tween 20. Two-fold linear dilution series(0.078–2.5 mM) of CIP in running buffer were passed over the ligand at the flowrate of 45 ml/min and all the sensorgrams were recorded at 22˚C. Surface re-generation was achieved by injecting a solution of 25 mM NaOH. Sensorgrams

2 CIP INTERFERES WITH CLASSICAL AND LECTIN COMPLEMENT PATHWAYS

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from three sets of data for each concentration were collected. Association anddissociation kinetics parameters (Ka and Kd) and the equilibrium dissociationconstant KD were estimated with a 1:1 interaction model (Langmuir model) bynonlinear fitting, using BIAevaluation 1.0 software.

ELISA to investigate complement deposition on GBS

Deposition of C3b from human serum on the streptococcal surface. GBS2603 (5 3 107 CFU) grown to stationary phase were coated onto microtiterwells overnight at 37˚C in 50 mM carbonate buffer (pH 9.5). The wells werewashed, blocked with 2% BSA for 1 h at 22˚C, and incubated for 10 min at

22˚C with 100 ml of normal or C1q-depleted human sera diluted to 5% with20 mM HEPES (pH 7.4) containing 140 mM NaCl, 2 mM CaCl2, 2 mMMgCl2, and 0.1% (w/v) BSA, or with 20 mM HEPES (pH 7.4) containing140 mM NaCl, 4 mM MgCl2, 16 mM EGTA, and 0.1% (w/v) BSA andpreincubated with increasing amounts (0–6 mM) of CIP. After washing, C3bdeposition was detected by rabbit anti-C3 polyclonal Ab (1:2000) followedby HRP-conjugated goat anti-rabbit IgG (1:1000).

Deposition of purified C3b and C4b on the streptococcal surface. GBS2603 (5 3 107 CFU) was immobilized as above. Wells were washed,blocked with 2% BSA for 1 h at 22˚C, and mixed with purified C3b or C4b

FIGURE 2. The CIP protein interferes with

the classical and lectin complement pathways,

but not with the AP. Human sera were pre-

incubated with CIP or an unrelated control

protein (Fib3) and loaded on ELISA wells

coated with LPS, IgM, or mannan. In (A),

plates were subsequently incubated with alka-

line phosphatase–labeled anti-C5b-9 Ab, whereas

anti-C3 or anti-C4 Ab and HRP-conjugated

secondary Abs were used in (B). The negative

control consisted of heat-inactivated serum. (C)

Effect of CIP on the inhibition of the classical

complement pathway. The effect of CIP (87.5–

350 nM) or Fib3 (350 nM) was examined by

measuring complement-mediated lysis of Ab-

coated sheep RBCs after serum preincubation

with the two proteins. The data are expressed

as percentage lysis compared with non-pre-

incubated serum. The reported data are the

mean values from three independent experi-

ments. Statistically significant differences are

indicated (*p , 0.05, ***p , 0.001) as deter-

mined by repeated-measures two-way ANOVA

with a Bonferroni posttest.

FIGURE 1. Amino acid sequence comparison of CIP from GBS strain COH1 with Eap (A) and Efb (B) from S. aureus strains 8325 and COL, respectively.

Conserved amino acids are indicated in color.

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(1 mg) preincubated with increasing concentrations of CIP (0–6 mM) in 20mM HEPES (pH 7.4) with 140 mM NaCl, 2 mM CaCl2, 2 mMMgCl2, and0.1% (w/v) BSA (assay buffer) in a total volume of 100 ml. After incubationfor 10 min, unbound components were washed away with assay buffer, anddeposited C3b or C4b was detected by rabbit anti-C3 or goat anti-C4polyclonal Abs (1:2000) and HRP-conjugated secondary Abs (1:1000).

Detection of CIP on culture supernatants and the GBS surface

GBS bacteria were grown to stationary phase in M9 medium and centri-fuged at 1500 3 g. One milliliter culture medium was directly adsorbedonto a nitrocellulose membrane (dot blot) or 5% TCA precipitated andloaded onto SDS-PAGE followed by Western blot. Detection of CIP wasperformed as indicated in the SDS-PAGE and Western blotting section.

To evaluate the presence of CIP on the surface of GBS, 5 3 107 CFUGBS grown to stationary phase were coated overnight onto microtiter wellsat 37˚C in 50 mM carbonate buffer (pH 9.5). Immobilized GBS was an-alyzed for the presence of CIP by addition of rabbit anti-CIP polyclonalsera (1:2000) followed by HRP-conjugated goat anti-rabbit IgG (1:1000).To examine binding of externally added CIP to the surface of GBS, bac-teria were immobilized onto microtiter wells and then incubated with 1 mgpurified recombinant CIP for 1 h at 22˚C. The wells were washed and thetotal amount of CIP bound to plates determined as reported above.

Whole-blood survival assays

The ability of GBS to survive in human blood in the presence of 2.5 or5 mM CIP was measured as previously described (25). Briefly, S. agalactiaestrains 6313 and 2603 grown to exponential phase (OD600 of 0.3) werediluted in Todd–Hewitt broth, and 100 ml (∼5 3 103 CFU) was added to0.9 ml fresh blood obtained from human healthy volunteers and treatedwith 50 mg/ml of the anticoagulant hirudin (Refludan), (Pharmion, Rome,Italy). In control experiments, cytochalasin (10 mg/ml) was added to thereaction. Tubes were incubated at 37˚C with gentle rocking and after 3 h,serial dilutions were plated to determine the number of surviving CFU. In asecond set of control experiments, the growth of each strain in humanplasma was checked in the presence of 5 mM CIP.

ELISA determination of anti-GBS IgG in human sera

GBS bacteria (5 3 107 CFU) grown to stationary phase or GBS polysac-charides Vor III conjugated to HSA (100 ng), prepared as described by Niloet al. (26), were coated overnight onto microtiter wells at 37˚C in 50 mMcarbonate buffer and then washed three times in washing buffer (0.05%Tween 20 in PBS). Plates were added with human sera diluted in PBS, 2%BSA, 0.05% Tween 20, incubated at 37˚C for 1 h, washed with 0.05% Tween20 in PBS, and then incubated for 90 min at 37˚C with alkaline phosphatase–conjugated anti-human IgG (Sigma-Aldrich) in PBS/2% BSA/0.05%Tween 20. After washing, the plates were developed with para-NitrophenylPhosphate (4 mg/ml) and the absorbance was measured at 405 nm. Stan-dard sera with known anti-polysaccharides III and V IgG concentrationswere obtained from Baylor College (Houston, TX) (27). Based on theseassays, the low titer sera used in our experiments contained,0.5 mg/ml anti-capsular Abs and yielded OD values ,0.3 in ELISA with whole bacteria.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism statistical analysissoftware. Differences between groups were analyzed by ANOVA with theappropriate posttest and by using repeated measures where required. A pvalue ,0.05 was considered statistically significant.

ResultsIdentification of CIP, a GBS protein that interferes with theclassical and lectin complement pathways

In the attempt to identify GBS-secreted virulence effectors mediat-ing host complement evasion, we interrogated a library of surface-predicted Ags (28) for the possible presence of proteins devoid oftransmembrane or leucine-proline-any-threonine-glycine motifs thatcould interfere with the activation of one or more complementpathways. We focused our attention on a 153-residue polypeptideshowing a partial homology with the staphylococcal secreted com-plement inhibitors Efb and Eap (∼15% identity and 35% similarityto both proteins) (Fig. 1). The corresponding gene was first annotatedas san_2130 (now COH1_1804) in the genome sequence of the GBSserotype III ST-17 strain COH1. The COH1 open reading frame

lacking the predicted hydrophobic leader region was cloned into aE. coli high copy number plasmid under a strong promoter. A sol-uble His-tagged protein of ∼15 kDa was purified with high yieldsfrom a positive clone.The SAN_2130 recombinant protein was tested for its ability to

inhibit the human complement pathways, using the Wielisa kit thatmeasures complement activation in human serum by an Ab directedagainst the C5b-9 terminal complex (24). In the assay, specificactivation of the CP, AP, or LP is achieved by coating ELISAwellseither with IgM, LPS, or mannan, respectively. As shown inFig. 2A, preincubation of human serum with increasing amountsof SAN_2130 resulted in a dose-dependent inhibition of the CPand LP, as detected by a decreased deposition of the C5b-9complex. Inhibition levels reached those obtained when the as-say was performed with heat-inactivated serum. None of thecomplement pathways was affected by the presence of the unre-lated GBS protein Fib3, used as negative control (23).To assess whether any intermediate steps of the complement

activation process could be inhibited by SAN_2130, a variant of the

FIGURE 3. CIP forms a complex with C4. (A) Western blot analysis of

C4 binding to CIP. Affinity-purified His-tagged CIP was subjected to

12.5% SDS-PAGE (5 mg/lane), electroblotted onto a nitrocellulose mem-

brane, and incubated with 1% normal (lane 3) or C4-depleted human se-

rum (lane 4). The membrane was probed with goat anti-C4 serum and

HRP-conjugated anti-goat Abs. Lane 1 shows a reference sample of CIP

subjected to SDS-PAGE and Coomassie blue staining. In lane 2, CIP

transferred to nitrocellulose membrane was detected by rabbit anti-CIPAb.

Molecular mass markers are indicated (kilodaltons). (B) Dose-dependent

binding of C4 to surface-coated CIP. Microtiter wells were coated with 100

ng CIP/well. The wells were probed with serial dilutions of normal human

serum, followed by incubation with goat anti-C4 and HRP-conjugated anti-

goat IgG. The graph is representative of three experiments with each point

indicating the average of triplicate wells.

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Wielisa assay where the presence of C3b or C4b on the platesurface was detected with appropriate specific antisera was set up.In the new conditions, preincubation of human serum withSAN_2130 resulted in a strong reduction of C3b detection in thewells where the CP and LP were tested, whereas no effect on C4bdeposition was observed for any of the pathways. As expected,SAN_2130 failed to block deposition of C3b via AP (Fig. 2B).The complement inhibitory effect of SAN_2130 was further

investigated using the CH50 assay, where activation of the clas-sical complement pathway can be examined by measuring hemo-globin release by a standardized suspension of sheep erythrocytessensitized with specific Abs. Upon addition of SAN_2130, a dose-dependent reduction of erythrocyte lysis was observed, whereas thecontrol protein Fib3 did not show any effect (Fig. 2C).The obtained data revealed an inhibitory effect of SAN_2130 on

human complement activation via CP and LP, and for this reasonthe protein was named CIP.

CIP shows high affinity for the C4 complement component

The functional studies presented above suggested that CIP inhibitedan event mediating activation of C3 via the CP and the LP. Wepredicted that CIP could act on either the fully assembled CP/LPC3 convertase (C4b2a) or an isolated component thereof. In aWestern blot assay, CIP bound to C4 present in EDTA-treatedserum, whereas no signal was visualized when a C4-containingmembrane was overlaid with a C4-depleted serum (Fig. 3A).This result was confirmed by an ELISA in which C4 from humanserum was demonstrated to bind dose-dependently to surface-coated CIP (Fig. 3B). A similar saturable binding was obtainedwhen purified C4 or C4b was immobilized on microtiter wellsand incubated with soluble CIP, whereas no significant interactionwas detected when increasing concentrations of CIP were incu-bated with immobilized purified C2 (Fig. 4A). To determine theaffinity of the CIP interaction with C4b, a SPR study was conducted.

Human C4b immobilized on the surface of a dextran chip wasincubated with soluble CIP concentrations ranging from 0.078 to2.5 mM. The GBS protein bound to C4b in a dose-dependentmanner, with a measured apparent KD of 95.36 5.2 nM (Fig. 4B).

CIP interferes with the initial C4b/C2 interaction

Formation of the CP/LP C3 convertase is a stepwise process thatstarts with the deposition of surface-bound C4b. Although C4b hasno enzymatic activity on its own, it serves as a molecular scaffoldbinding to C2 to yield the C4bC2 proconvertase and for C1s/MASP-dependent cleavage of C2 to generate the fully activeC4b2a convertase. Therefore, we examined the effect of CIP on theinteraction between purified C4b and C2 in an ELISA format.As reported in Fig. 5A, CIP inhibited binding of C4b to surface-

coated C2 in a dose-dependent fashion. Conversely, no interferingeffect by CIP was observed on the binding of Bb to immobilizedC3b (Fig. 5B). Taken together, these data suggested that binding ofCIP to C4b constitutes the molecular basis of the specific inhibitionof both CP and LP activity and confirmed that the streptococcalprotein does not affect the formation of the C3bBb convertase.

CIP is expressed by a subset of GBS human and bovine strains

We subsequently examined the expression and localization of theCIP protein across eight sequenced GBS strains of human or bo-vine origin (22), six of which contain the cip open reading framein their genome.To assess secretion of the CIP protein, bacteria were grown to

stationary phase and culture supernatants were spotted onto anitrocellulose membrane, followed by incubation with anti-CIPsera. As shown in Fig. 6A, different amounts of protein weresecreted by all the strains carrying the gene, whereas no signal wasdetected on the spots corresponding to the two cip2 isolates.Conversely, the unrelated and highly conserved GBS protein Sipwas uniformly expressed in all the strains tested. The specificity of

FIGURE 4. CIP binds purified C4b in a dose-de-

pendent manner and with high affinity. (A) Increasing

amounts of CIP were added to wells coated with pu-

rified C4b or C2 (500 ng/well). Bound protein was

detected by addition of rabbit anti-CIP IgG, followed

by HRP-conjugated anti-rabbit IgG. (B) SPR analysis

of CIP binding to C4b. Two-fold linear dilution series

(0.078–2.5 mM) of CIP were injected over the C4b

surface (250 response units) on a sensor chip CM5

and the sensorgrams were recorded at pH 7.4 and

25˚C. The representative sensorgrams have been cor-

rected for the response obtained when the recombinant

GBS protein was flowed over uncoated chips. Sensor-

grams are shown in black (experimental data) and in

red (fitted curves). The figure shows one representative

of three experiments. Kinetics constants and results of

statistical analysis are reported in the inset.

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the dot blot was further confirmed by a Western immunoblottingassay, where a protein with a molecular mass corresponding to CIPwas detected in the supernatants of cip+ but not in cip2 supernatants(Fig. 6B). Remarkably, all the above reported results were obtainedwhen a chemically defined medium added with 0.3% yeast extractwas used for GBS growth, whereas no expression of CIP was de-tected using Todd–Hewitt broth or other rich media (unpublishedresults), suggesting tight regulation of CIP expression.To investigate whether CIP could also be found on the GBS

surface, bacteria grown to stationary phase were directly spottedonto ELISA microtiter wells, followed by incubation with anti-CIPand HRP-conjugated secondary Abs. As shown in Fig. 6C, thestrains containing the cip gene responded positively to the Abs,whereas no signal was detected on the cip2 bacteria. Notably,externally added CIP to immobilized strains resulted in highlyincreased signals, indicative of the protein binding to the surfaceof the bacteria. CIP bound to all GBS strains irrespective of thepresence of the corresponding gene in their genome, suggestingthat capturing of CIP is an intrinsic property of the GBS surface.Overall, the data suggested that CIP is secreted by the bacteria andcan rebind to the GBS surface.

CIP inhibits C3b deposition on the GBS surface by blockingthe formation of C3b via the C4bC2a convertase

We sought to determine whether the interaction of CIP with C4could have an impact on the deposition of C3b from human serumon the GBS cell surface, possibly by inhibiting the formation of the

CP/LP C4b2a convertase. This was examined incubating the type VGBS 2603 strain lacking the cip gene, with human serum in thepresence of increasing concentrations of CIP. For these experi-ments we used a normal human serum containing low levels ofAbs against GBS 2603, as assessed by ELISA.The serum was diluted with Mg2+/Ca2+- or Mg2+/EGTA-

containing buffers, preincubated with increasing amounts of CIPor with buffer alone, and the mixtures were added to microtiterwells coated with GBS 2603. After washing, the plates were ex-amined for the presence of C3b bound to bacteria with anti-C3IgG. As shown in Fig. 7A, in the absence of CIP, OD values of∼0.7 were detected when only the AP was operational (buffercontaining Mg2+/EGTA), whereas OD saturating values of 2.5

FIGURE 5. CIP interferes with the initial C4b/C2 interaction. (A) Effect

of CIP on the binding of C4b to surface-coated C2. Microtiter wells coated

with C2 (500 ng/well) were incubated with 1 mg purified human C4b in the

presence of increasing concentrations of CIP. Inhibition of C4b binding to

C2 is expressed as percentage of that observed in the absence of inhibitor.

Error bars represent means 6 SD of three independent experiments per-

formed in triplicate. (B) The same experiment as in (A) except that inhi-

bition by CIP of C3b (1 mg/well) binding to immobilized factor B (500 ng/

well) in the presence of 5 mM Mg2+ was investigated.

FIGURE 6. CIP is secreted by a subset of GBS isolates and captured on

the bacterial surface. (A) Dot blot analysis of bacterial culture supernatants

incubated with rabbit anti-CIP or mouse anti-Sip and HRP-labeled sec-

ondary Abs. (B) Western blot analysis of TCA-precipitated culture su-

pernatants incubated with rabbit anti-CIP and HRP-labeled goat Abs. (C)

Binding of endogenous or externally added CIP to GBS coated onto

microtiter wells was determined by addition of rabbit anti-CIP and sec-

ondary anti-rabbit Abs. Statistically significant differences are indicated

(**p, 0.01) as determined by repeated-measures two-way ANOVAwith a

Bonferroni posttest.

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were measured with serum containing Mg2+/Ca2+ ions. This find-ing was consistent with previous reports indicating that, in theabsence of specific Abs, GBS is mainly susceptible to the classicaland/or the lectin pathways, whereas its sialic acid–containingcapsule renders the organism resistant to the complement AP (6).Remarkably, addition of increasing concentrations of CIP inMg2+/Ca2+ buffer progressively reduced C3b deposition to levelscomparable to those observed when serum was incubated withMg2+/EGTA, either in the absence or presence of CIP (Fig. 7A). Insummary, CIP appeared to inhibit C3b deposition via the classicaland/or the lectin complement pathways without affecting the AP.To understand which of the two pathways (CP or LP) was more

relevant in the explored experimental setting, we tested the po-tential activity of CIP in the same assay using a C1q-depleted lowanti-GBS Ab titer serum. The results were equivalent to thosereported in Fig. 7A in terms both of C3b deposition in Mg2+/EGTA- or Mg2+/Ca2+-containing buffers, and of CIP-promotedinhibition levels (Fig. 7B). These data pointed toward a key roleof the LP in C3b deposition on the GBS surface in the absence ofspecific Abs, and confirmed the interfering effect of CIP on the LPactivation.To distinguish whether the observed CIP effect was a conse-

quence of the inhibition of C3b formation by the LP C4bC2aconvertase rather than the result of a direct interference on C3bdeposition, purified C3b was preincubated with increasing amountsof soluble CIP and the mixture was added to wells coated with GBS2603. As shown in Fig. 8A, the effect of soluble CIP on bacterialdeposition of C3b was negligible even at the highest concentra-

tions. A similar observation was made when the CIP effect on C4bdeposition was examined.Expanding this analysis, CIP was first preincubated with

immobilized GBS 2603 to allow binding of the protein to thebacterial surface, followed by addition of C3b or C4b. No decreasein C3b/C4b deposition was detected either of these new experi-mental conditions (Fig. 8B).Together with the former observation that CIP blocks the in-

teraction of the purified C4b with C2, these results definitivelyconfirm that CIP interferes with C3b formation via the C4b2aconvertase, rather than preventing C3b or C4b deposition.

CIP inhibits GBS killing in a whole-blood assay

To gain additional insights on the involvement of CIP in GBSpathogenesis, experiments were designed aimed at assessing theeffect of CIP on bacterial survival in human whole blood in theabsence of anti-capsular Abs. Blood samples from selected donorswith very low anti-GBS Ab titer were used for this analysis.Recombinant hirudin was used as anticoagulant to preservecomplement activity. A dose-dependent inhibition of killing of thecip2 GBS strains 6313 (Fig. 9A) and 2603 (Fig. 9B) in thepresence of 2.5 or 5 mM CIP was observed, whereas no effect wasdetected with the protein Fib3. In a control experiment, bacteriawere incubated for 3 h in blood and added with cytochalasin D toblock phagocytosis. The treatment almost completely abolishedphagocytosis, demonstrating the involvement of actin cytoskeletalrearrangements in the process. To further exclude the possibility

FIGURE 7. Impact of recombinant CIP on C3b deposition on the GBS

surface. Normal (A) or C1q-depleted human sera (B) containing low levels

of anti-GBS Abs were diluted to 5% with Mg2+/Ca2+- or Mg2+/EGTA-

containing buffer and preincubated with increasing amounts of CIP. The

mixtures were added to microtiter wells coated with GBS 2603. After

incubation, deposition of C3b was detected by addition of a rabbit anti-C3

IgG followed by a HRP-conjugated anti-rabbit IgG. Error bars represent

means 6 SD of three independent experiments.

FIGURE 8. Binding of purified C3b or C4b to the GBS surface in

presence of CIP. (A) C3b or C4b was preincubated with increasing

amounts of CIP and then added to microtiter wells coated with GBS 2603.

Following incubation, C3b and C4b deposition was detected by using

specific Abs. (B) GBS 2603 was surface coated to microtiter wells and then

allowed to incubate and adsorb the indicated concentrations of CIP. The

wells were added with C3b or C4b and after incubation the amounts of the

bound complement components determined as reported in (A). Error bars

represent means 6 SD of three independent experiments.

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that the observed CIP effect could be somehow related to the al-teration of GBS growth, streptococci were grown in plasma (ab-sence of blood cells) in the presence/absence of CIP. In bothconditions GBS proliferated and grew to the same extent (data notshown). These experiments indicated that CIP can play a role inthe ability of GBS to survive in human blood by preventingphagocytosis.

DiscussionThe effective uptake and killing of GBS by host phagocytic cellsrequire opsonization of the bacterium by specific Abs and/or thecomplement system. To date most of the work describing the in-teractions between S. agalactiae and complement componentshas primarily focused on the role of sialic acid–rich capsule (6)and factor H–binding proteins (12, 29) in the prevention of C3-mediated opsonization through inhibition of the AP. Low levels ofmaternal anti-capsular polysaccharide IgG correlate with an in-creased neonatal susceptibility to GBS infection (30), and a higheramount of these Abs increases the efficiency of GBS killing bypolymorphonuclear phagocytes via a complement-dependentmechanism involving the AP (31, 32).Serum deficient in anti-capsular IgG can still trigger significant

phagocytic killing of GBS by a process involving complementactivation that requires Ca2+ and Mg2+ and is hence AP-independent

(32, 33). It is therefore presumable that these bacteria have evolvedadditional virulence factors counteracting the activation of the CPor LP.In this study, we describe, to our knowledge for the first time, a

proteinaceous factor secreted in the culture medium of GBS,which we named CIP, that forms a high-affinity complex with C4band can block the assembly of the CP/LP C4bC2a proconvertase,resulting in impaired C3b deposition. In fact, CIP bound C4b in asaturable manner with high affinity. The C4bC2a inhibitory effectof CIP was highly specific, as there was no interference on thebinding of Bb to immobilized C3b (AP convertase), nor on thedeposition of purified C3b and C4b on the GBS surface.Even though CIP could in principle hamper both CP and LP

activation, when we tested its effect in sera with low titers of anti-GBS IgG, we observed inhibition of the formation/deposition ofC3b irrespective of the presence of C1q. The data confirm previousobservations indicating that C1q binding to the GBS surface (34) isper se not sufficient to mediate Ab-independent opsonophagocytickilling (35). Recent studies highlighted a role of the LP L-ficolin/MASP complexes in the formation of the C4bC2a convertase onthe GBS surface, initiation of C3b deposition, and phagocytickilling in the absence of specific Abs (36), a process that can befurther amplified by anti-capule polysaccaride IgG and the APconvertase. The same authors suggested that deficiencies inL-ficolin in cord serum could be a risk factor for neonatal GBSinfection (37). We concluded that, in absence of specific Abs, CIPcould interfere with LP complement amplification. Interestingly,Ali et al. (38) recently demonstrated that the LP L-ficolin is also acritical component of the innate immune response to pneumo-coccal infection.Recent preliminary evidence indicates that CIP can also bind

C3b (unpublished results). However, as reported in Figs. 2 and 5B,this interaction does not prevent the formation of the AP con-vertase, nor C3b deposition on the bacterial surface via AP (Fig. 6).The biochemical characterization of CIP binding to C3b and thebiological implications of this new CIP interaction are currentlyunder investigation in our laboratories.Searching for CIP homologs in other bacterial species, we

found that a subset of strains from another human streptococcalpathogen, the group A Streptococcus (GAS), expresses a proteindisplaying 46% identity with CIP. The GBS cip gene and itsGAS homolog are present in a similar phage-derived genomicregion named RD2 that was acquired by horizontal transfer andis integrated into a tRNA gene flanked by direct repeats. Inaddition to CIP, RD2 encodes several proteins with predictedsecretion signal sequences, among which the R28 protein thathas been implicated in host–pathogen interactions (39). Inter-estingly, in GAS this region is present in all serotype M28strains and in strains of other serotypes associated with mater-nal–fetal urogenital infections (40, 41). In the case of GBS,analysis of the 373 genomes present in the NCBI databaserevealed 80 cip+ strains mainly belonging to the hypervirulenttype III ST-17, ST-23, and to the bovine ST-61-67.Although CIP is secreted, the purified soluble protein can bind

to the bacterial surface. Rebinding of CIP to GBS occurs both oncip+ and cip2 strains, suggesting that the GBS cell wall peptido-glycan contains yet undetermined components that can recognizeand bind to CIP. Thus, we speculate that in coculture conditionscip+ GBS strains can transfer to cip2 strains the ability to neutralizecomplement system.The mechanism by which CIP interferes with CP and LP acti-

vation is reminiscent of the recently described effect of the Eap-secreted protein expressed by S. aureus. Indeed, Woehl et al.(17) demonstrated a direct nanomolar affinity interaction of Eap

FIGURE 9. CIP interferes with GBS killing in a whole-blood assay.

S. agalactiae 6313 (A) and 2603 (B) were tested for their ability to survive

after 3 h of incubation in human blood containing low anti-GBS IgG.

Surviving bacteria were detected by viable counting. Presented data are

the mean killing percentage 6 SD of three experiments using blood

from independent donors. Statistically significant differences are indicated

(*p , 0.05, **p , 0.01) as determined by repeated-measures two-way

ANOVA with a Bonferroni posttest.

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with C4b and consequent inhibition of C4bC2 assembly. Thesimilarities between CIP and Eap go beyond the mechanism bywhich they disrupt the formation of the CP/LP proconvertase. Infact, CIP and Eap display partial homology, both are secreted inthe culture medium and rebind to the bacterial surface, and theyultimately interfere with the deposition of C3b and phagocytickilling.The contribution of CIP to GBS virulence by using isogenic

mutant strains remains to be investigated. Woehl et al. (17) ob-served that a knockout mutant carrying an in-frame deletion ofEap did not show reduced levels of bacterial C3b deposition orphagocytosis compared with wild-type, whereas a protective ef-fect was achieved when the protein was added exogenously, as weobserved for GBS CIP. The data suggested that exogenous Eap,but not surface-retained Eap, could significantly contribute toS. aureus complement evasion.In conclusion, the results obtained in our studies provide new

insights into the mechanisms of GBS immune evasion and uncovera novel strategy of CP/LP regulation that may hold significantimplications for prophylactic/therapeutic interventions againstoverwhelming infections caused by this pathogen.

AcknowledgmentsWe acknowledge Monica Fabbrini and Alessandra Acquaviva for ELISA

experiments to determine anti-GBS titers in the human sera used for our

study and Francesco Berti, Barbara Brogioni, and Pala Lo Surdo for support

on SPR experiments. We thank the investigators who provided the GBS

strains ES-NI-010 (Javier Rodriguez, DEVANI program, European Com-

mission Seventh Framework no. 200481), 2603 (Graziella Orefici, ISS,

Rome, Italy), COH1 (Dennis Kasper, Brigham and Women’s Hospital, Boston,

MA), 515 (Carol Baker, Baylor College of Medicine, Houston, TX), and

SH0248 and 383728 (Mario Rodriguez, University of Lisbon, Lisbon, Portugal).

DisclosuresI.M., R.R., and S.B. are employees of GSK Vaccines S.r.l. The remaining

authors have no financial conflicts of interest.

References1. Herbert, M. A., C. J. Beveridge, and N. J. Saunders. 2004. Bacterial virulence

factors in neonatal sepsis: group B streptococcus. Curr. Opin. Infect. Dis. 17:225–229.

2. Edwards, M. S., and V. Nizet. 2011. Group B streptococcal infections. In In-fectious diseases of the fetus and newborn infant, 7th Ed., J. S. Remington, andJ. O. Klein, eds. Elsevier Saunders, Philadelphia, p. 419–469. doi:10.1016/B978-1-4160-6400-8.00012-2

3. Phares, C. R., R. Lynfield, M. M. Farley, J. Mohle-Boetani, L. H. Harrison,S. Petit, A. S. Craig, W. Schaffner, S. M. Zansky, K. Gershman, et al; ActiveBacterial Core surveillance/Emerging Infections Program Network. 2008. Epi-demiology of invasive group B streptococcal disease in the United States, 1999–2005. JAMA 299: 2056–2065.

4. Berends, E. T., A. Kuipers, M. M. Ravesloot, R. T. Urbanus, andS. H. Rooijakkers. 2014. Bacteria under stress by complement and coagulation.FEMS Microbiol. Rev. 38: 1146–1171.

5. Berti, F., E. Campisi, C. Toniolo, L. Morelli, S. Crotti, R. Rosini, M. R. Romano,V. Pinto, B. Brogioni, G. Torricelli, et al. 2014. Structure of the type IX group BStreptococcus capsular polysaccharide and its evolutionary relationship withtypes V and VII. J. Biol. Chem. 289: 23437–23448.

6. Edwards, M. S., D. L. Kasper, H. J. Jennings, C. J. Baker, and A. Nicholson-Weller. 1982. Capsular sialic acid prevents activation of the alternative com-plement pathway by type III, group B streptococci. J. Immunol. 128: 1278–1283.

7. Baker, C. J., B. J. Webb, D. L. Kasper, and M. S. Edwards. 1986. The role ofcomplement and antibody in opsonophagocytosis of type II group B strepto-cocci. J. Infect. Dis. 154: 47–54.

8. Wessels, M. R., C. E. Rubens, V. J. Benedı, and D. L. Kasper. 1989. Definition ofa bacterial virulence factor: sialylation of the group B streptococcal capsule.Proc. Natl. Acad. Sci. USA 86: 8983–8987.

9. Marques, M. B., D. L. Kasper, M. K. Pangburn, and M. R. Wessels. 1992.Prevention of C3 deposition by capsular polysaccharide is a virulence mecha-nism of type III group B streptococci. Infect. Immun. 60: 3986–3993.

10. Areschoug, T., M. Stalhammar-Carlemalm, I. Karlsson, and G. Lindahl. 2002.Streptococcal b protein has separate binding sites for human factor H and IgA-Fc. J. Biol. Chem. 277: 12642–12648.

11. Carlin, A. F., Y. C. Chang, T. Areschoug, G. Lindahl, N. Hurtado-Ziola,C. C. King, A. Varki, and V. Nizet. 2009. Group B Streptococcus suppression ofphagocyte functions by protein-mediated engagement of human Siglec-5. J. Exp.Med. 206: 1691–1699.

12. Maruvada, R., N. V. Prasadarao, and C. E. Rubens. 2009. Acquisition of factor Hby a novel surface protein on group B Streptococcus promotes complementdegradation. FASEB J. 23: 3967–3977.

13. Santi, I., M. Scarselli, M. Mariani, A. Pezzicoli, V. Masignani, A. Taddei,G. Grandi, J. L. Telford, and M. Soriani. 2007. BibA: a novel immunogenicbacterial adhesin contributing to group B Streptococcus survival in human blood.Mol. Microbiol. 63: 754–767.

14. Rooijakkers, S. H., F. J. Milder, B. W. Bardoel, M. Ruyken, J. A. van Strijp, andP. Gros. 2007. Staphylococcal complement inhibitor: structure and active sites. J.Immunol. 179: 2989–2998.

15. Lee, L. Y., M. Hook, D. Haviland, R. A. Wetsel, E. O. Yonter, P. Syribeys,J. Vernachio, and E. L. Brown. 2004. Inhibition of complement activation by asecreted Staphylococcus aureus protein. J. Infect. Dis. 190: 571–579.

16. Lee, L. Y., X. Liang, M. Hook, and E. L. Brown. 2004. Identification andcharacterization of the C3 binding domain of the Staphylococcus aureus extra-cellular fibrinogen-binding protein (Efb). J. Biol. Chem. 279: 50710–50716.

17. Woehl, J. L., D. A. Stapels, B. L. Garcia, K. X. Ramyar, A. Keightley,M. Ruyken, M. Syriga, G. Sfyroera, A. B. Weber, M. Zolkiewski, et al. 2014.The extracellular adherence protein from Staphylococcus aureus inhibits theclassical and lectin pathways of complement by blocking formation of the C3proconvertase. J. Immunol. 193: 6161–6171.

18. Wilson, C. B., and W. M. Weaver. 1985. Comparative susceptibility of group Bstreptococci and Staphylococcus aureus to killing by oxygen metabolites. J.Infect. Dis. 152: 323–329.

19. Wessels, M. R., L. C. Paoletti, A. K. Rodewald, F. Michon, J. DiFabio,H. J. Jennings, and D. L. Kasper. 1993. Stimulation of protective antibodiesagainst type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanustoxoid conjugate vaccine. Infect. Immun. 61: 4760–4766.

20. Tettelin, H., V. Masignani, M. J. Cieslewicz, J. A. Eisen, S. Peterson,M. R. Wessels, I. T. Paulsen, K. E. Nelson, I. Margarit, T. D. Read, et al. 2002.Complete genome sequence and comparative genomic analysis of an emerginghuman pathogen, serotype V Streptococcus agalactiae. Proc. Natl. Acad. Sci.USA 99: 12391–12396.

21. Samen, U., B. Gottschalk, B. J. Eikmanns, and D. J. Reinscheid. 2004. Rele-vance of peptide uptake systems to the physiology and virulence of Strepto-coccus agalactiae. J. Bacteriol. 186: 1398–1408.

22. Rosini, R., E. Campisi, M. De Chiara, H. Tettelin, D. Rinaudo, C. Toniolo,M. Metruccio, S. Guidotti, U. B. Skov Sørensen, M. Kilian, et al.2015. Genomicanalysis reveals the molecular basis for capsule loss in the group B Streptococcuspopulation. PLoS One 10: e0125985 doi:10.1371/journal.pone.0125985.

23. Margarit, I., S. Bonacci, G. Pietrocola, S. Rindi, C. Ghezzo, M. Bombaci,V. Nardi-Dei, R. Grifantini, P. Speziale, and G. Grandi. 2009. Capturing host-pathogen interactions by protein microarrays: identification of novel strepto-coccal proteins binding to human fibronectin, fibrinogen, and C4BP. FASEB J.23: 3100–3112.

24. Seelen, M. A., A. Roos, J. Wieslander, T. E. Mollnes, A. G. Sjoholm,R. Wurzner, M. Loos, F. Tedesco, R. B. Sim, P. Garred, et al. 2005. Functionalanalysis of the classical, alternative, and MBL pathways of the complementsystem: standardization and validation of a simple ELISA. J. Immunol. Methods296: 187–198.

25. Visai, L., N. Yanagisawa, E. Josefsson, A. Tarkowski, I. Pezzali,S. H. Rooijakkers, T. J. Foster, and P. Speziale. 2009. Immune evasion byStaphylococcus aureus conferred by iron-regulated surface determinant proteinIsdH. Microbiology 155: 667–679.

26. Nilo, A., L. Morelli, I. Passalacqua, B. Brogioni, M. Allan, F. Carboni,A. Pezzicoli, F. Zerbini, D. Maione, M. Fabbrini, et al. 2015. Anti-group BStreptococcus glycan-conjugate vaccines using pilus protein GBS80 as carrierand antigen: comparing lysine and tyrosine-directed conjugation. ACS Chem.Biol. 10: 1737–1746.

27. Guttormsen, H. K., C. J. Baker, M. S. Edwards, L. C. Paoletti, and D. L. Kasper.1996. Quantitative determination of antibodies to type III group B streptococcalpolysaccharide. J. Infect. Dis. 173: 142–150.

28. Maione, D., I. Margarit, C. D. Rinaudo, V. Masignani, M. Mora, M. Scarselli,H. Tettelin, C. Brettoni, E. T. Iacobini, R. Rosini, et al. 2005. Identification of auniversal group B Streptococcus vaccine by multiple genome screen. Science309: 148–150.

29. Jarva, H., J. Hellwage, T. S. Jokiranta, M. J. Lehtinen, P. F. Zipfel, and S. Meri.2004. The group B streptococcal b and pneumococcal Hic proteins are struc-turally related immune evasion molecules that bind the complement inhibitorfactor H in an analogous fashion. J. Immunol. 172: 3111–3118.

30. Baker, C. J., and D. L. Kasper. 1976. Correlation of maternal antibody deficiencywith susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med.294: 753–756.

31. Edwards, M. S., C. J. Baker, and D. L. Kasper. 1979. Opsonic specificity ofhuman antibody to the type III polysaccharide of group B Streptococcus. J.Infect. Dis. 140: 1004–1008.

32. Edwards, M. S., A. Nicholson-Weller, C. J. Baker, and D. L. Kasper. 1980. Therole of specific antibody in alternative complement pathway-mediated opsono-phagocytosis of type III, group B Streptococcus. J. Exp. Med. 151: 1275–1287.

33. Baker, C. J., M. S. Edwards, B. J. Webb, and D. L. Kasper. 1982. Antibody-independent classical pathway-mediated opsonophagocytosis of type Ia, group Bstreptococcus. J. Clin. Invest. 69: 394–404.

The Journal of Immunology 9

by guest on March 24, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

34. Levy, N. J., and D. L. Kasper. 1986. Surface-bound capsular polysaccharide of

type Ia group B Streptococcus mediates C1 binding and activation of the classic

complement pathway. J. Immunol. 136: 4157–4162.35. Butko, P., A. Nicholson-Weller, and M. R. Wessels. 1999. Role of complement

component C1q in the IgG-independent opsonophagocytosis of group B Strep-

tococcus. J. Immunol. 163: 2761–2768.36. Aoyagi, Y., E. E. Adderson, J. G. Min, M. Matsushita, T. Fujita, S. Takahashi,

Y. Okuwaki, and J. F. Bohnsack. 2005. Role of L-ficolin/mannose-binding lectin-

associated serine protease complexes in the opsonophagocytosis of type III

group B streptococci. J. Immunol. 174: 418–425.37. Fujieda, M., Y. Aoyagi, K. Matsubara, Y. Takeuchi, W. Fujimaki, M. Matsushita,

J. F. Bohnsack, and S. Takahashi. 2012. L-ficolin and capsular polysaccharide-

specific IgG in cord serum contribute synergistically to opsonophagocytic killing

of serotype III and V group B streptococci. Infect. Immun. 80: 2053–2060.

38. Ali, Y. M., N. J. Lynch, K. S. Haleem, T. Fujita, Y. Endo, S. Hansen,U. Holmskov, K. Takahashi, G. L. Stahl, T. Dudler, et al. 2012. The lectinpathway of complement activation is a critical component of the innate immuneresponse to pneumococcal infection. PLoS Pathog. 8: e1002793 .

39. Stalhammar-Carlemalm, M., T. Areschoug, C. Larsson, and G. Lindahl. 1999.The R28 protein of Streptococcus pyogenes is related to several group Bstreptococcal surface proteins, confers protective immunity and promotesbinding to human epithelial cells. Mol. Microbiol. 33: 208–219.

40. Green, N. M., S. Zhang, S. F. Porcella, M. J. Nagiec, K. D. Barbian, S. B. Beres,R. B. LeFebvre, and J. M. Musser. 2005. Genome sequence of a serotype M28strain of group a Streptococcus: potential new insights into puerperal sepsis andbacterial disease specificity. J. Infect. Dis. 192: 760–770.

41. Sitkiewicz, I., N. M. Green, N. Guo, L. Mereghetti, and J. M. Musser. 2011. Lateralgene transfer of streptococcal ICE element RD2 (region of difference 2) encodingsecreted proteins. BMC Microbiol. 11: 65 doi:10.1186/1471-2180-11-65.

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