BibA: a novel immunogenic bacterial adhesincontributing to group B Streptococcus survival inhuman blood

Isabella Santi,1 Maria Scarselli,1 Massimo Mariani,1

Alfredo Pezzicoli,1 Vega Masignani,1

Annarita Taddei,2 Guido Grandi,1 John L. Telford1

and Marco Soriani1*1Novartis Vaccines and Diagnostics Srl, Via Fiorentina1, 53100, Siena, Italy.2Centro Interdipartimentale Microscopia Elettronica,University of La Tuscia, Largo dell’Università, 01100,Viterbo, Italy.

Summary

By the analysis of the recently sequenced genomes ofGroup B Streptococcus (GBS) we have identified anovel immunogenic adhesin with anti-phagocyticactivity, named BibA. The bibA gene is present in100% of the 24 GBS strains analysed. BibA-specificIgG were found in human sera from normal healthydonors. The putative protein product is a polypeptideof 630 amino acids containing a helix-rich N-terminaldomain, a proline-rich region and a canonical LPXTGcell wall-anchoring domain. BibA is expressed on thesurface of several GBS strains, but is also recoveredin GBS culture supernatants. BibA specifically bindsto human C4-binding protein, a regulator of theclassic complement pathway. Deletion of the bibAgene severely reduced the capacity of GBS to survivein human blood and to resist opsonophagocytickilling by human neutrophils. In addition, BibAexpression increased the virulence of GBS in amouse infection model. The role of BibA in GBS adhe-sion was demonstrated by the impaired ability of abibA knockout mutant strain to adhere to both humancervical and lung epithelial cells. Furthermore, wecalculated that recombinant BibA bound to humanepithelial cells of distinct origin with an affinity con-stant of ~10-8 M for cervical epithelial cells. HenceBibA is a novel multifunctional protein involved inboth resistance to phagocytic killing and adhesion tohost cells. The identification of this potential new

virulence factor represents an important step in thedevelopment of strategies to combat GBS-associatedinfections.

Introduction

Group B Streptococcus (GBS) is the leading cause ofneonatal septicaemia and meningitis and it has recentlybeen recognized as an increasingly common cause ofinvasive disease in non-pregnant adults. Despite recentadvances in the elucidation of putative virulence traits,capsular polysaccharide is the only factor conclusivelylinked to GBS evasion of the immune system (Marqueset al., 1992; Jarva et al., 2003). Indeed, it is not yet knownwhether the Bac protein that has been reported to bind withhigh affinity to the Fc part of human serum IgA (Lindahlet al., 1990) and to complement regulator Factor H (FH)(Areschoug et al., 2002) has anti-phagocytic properties.By contrast, the sialic acid-rich capsule of GBS is known toprevent opsonophagocytosis and to avoid C3b deposition(Marques et al., 1992). In addition, asialylation of GBSpolysaccharide is associated with a diminished virulence(Edwards et al., 1982; Wessels et al., 1989).

On the other hand, a variety of information is availableon streptococcal survival strategies to host defences inGroup A Streptococcus (GAS) and Pneumococcus. Inparticular, GAS expresses a number of proteins, whichhave been postulated to have a crucial role in bacterialcolonization and anti-phagocytic activity. Such proteinsare characterized by their capacity to bind extracellularmatrix proteins, keratinocytes, immunoglobulins (Ig) andfluid-phase complement regulators (Thern et al., 1995;Darmstadt et al., 2000; Areschoug et al., 2004).

Group A Streptococcus acquires FH via M proteins andfibronectin-binding proteins, contributing to its capacity toevade phagocytosis by polymorphonuclear cells (Horst-mann et al., 1988; Pandiripally et al., 2002). M proteinsalso mediate acquisition of C4-binding protein (C4bp), animportant regulator of the classical complement pathwaycomponent C3 convertase (C4b2a) (Berggard et al.,2001). When bound to C4b, M protein has both decayaccelerating activity and cofactor activity for C4b cleavagein an analogous fashion to FH in the alternative pathway(Perez-Caballero et al., 2004; Carlsson et al., 2005).

Accepted 30 November, 2006. *For correspondence. E-mailmarco_soriani@chiron.com; Tel. (+39) 0577 245049; Fax(+39) 0577 243564.

Molecular Microbiology (2007) 63(3), 754–767 doi:10.1111/j.1365-2958.2006.05555.xFirst published online 8 January 2007

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd

Receptors for IgA and/or IgG also belong to the M proteinfamily (Stenberg et al., 1992). Of interest, GAS and GBSsecrete a common enzyme, the C5a peptidase, whichinactivates human C5a (Wexler et al., 1985) and bindsfibronectin, thereby promoting bacterial invasion of epi-thelial cells (Beckmann et al., 2002; Cheng et al., 2002).

We recently used a reverse vaccinology approach toidentify useful protein targets for the development of novelvaccines against GBS. To this end, the complete genomesof eight different GBS strains, representative of the majorvirulent serotypes, were analysed (Tettelin et al., 2005).Bioinformatic analyses of these strains led to the identifi-cation of a well-conserved, cell wall-anchored protein thatwe refer to as Group B Streptococcus immunogenic bac-terial adhesin (BibA). A BLAST search against the non-redundant GenBank database revealed some similarity ofBibA with streptococcal Ig-binding proteins such as the Mprotein family of GAS (Podbielski et al., 1994) and Hic ofStreptococcus pneumoniae (Dave et al., 2004). We per-formed in vitro biochemical and functional investigations ofBibA, revealing that the protein is able to bind human C4bp,as well as to promote the adhesion of GBS to humanepithelial cells. In addition, we showed that BibA is immu-nogenic in humans and confers resistance to phagocytickilling, contributing to GBS survival in human blood.

Results

BibA genomic characterization

Analysis of recently sequenced GBS genomes shows thatthe bibA gene is present in strains 2603 V/R (Tettelinet al., 2002), NEM316 type III (Glaser et al., 2002), COH1type III, CJB111 type V, 515 type Ia, 18RS21 type II andA909 type Ia (Tettelin et al., 2005), although interrupted bythe insertion of two putative transposases in A909. Thisinsertion causes the interruption of the reading frame atnucleotide 580. Genetic analysis from 25 strains, repre-senting the most common serotypes, revealed that bibAgene was present in 100% of the strains (Table 1). Inaddition, the BibA protein was exposed on the surface of58% of the strains; while in 24% of them BibA was exclu-sively recovered from the supernatants.

In general, two different types of sequence variabilitycan be observed among the BibA proteins. The first isthe presence of a variable number of brief amino acidmodules, which can be observed either within theN-terminal domain, or within the proline-rich tract. In par-ticular, the IKAESIN and KIQXKXNT repeats can beobserved within the N-terminal domain in A909, CJB111and H36B, while a varying number of copies of the PEAK/PDVK motif is present at the C-terminus of all the alleles.The second source of sequence variation consists of anon-repetitive tract of 97 residues within the proline-rich

region, which characterizes 515, NEM316, H36B, CJB111and A909 strains (Fig. 1).

Collectively, sequence analysis reveals that the proteinexists in four different variants: one found in strains 2603and 18RS21; another in NEM316 and 515; the third inCJB111, H36B and A909 and the fourth in COH1 (Fig. 1).Of interest, the COH1 variant was found to have highhomology to the bibA allele present in several bovinestrains (I. Santi, unpubl. results). The multiple alignment ofthe BibA amino acid sequences shows that the protein isgenerally well conserved. Amino acid identity rangesbetween 63.3% and 100% among N-terminal domains ofdifferent strains, with the exception of COH1, whoseN-terminal region shows on average about 25% aminoacid identity to the other alleles.

BibA is exposed on GBS surface

The putative protein product of the bibA gene in the GBSstrain 2603 V/R (Tettelin et al., 2002) is a polypeptide of630 amino acids containing a leader peptide (residues1–27), a N-terminal domain (residues 28–400), a proline-rich region (residues 401–568) that consists of 42 copiesof a PEAK/PDVK motif, and a canonical cell wall-anchoring domain (residues 596–630). The anchoringdomain is formed by the consensus LPXTG sequence,followed by a hydrophobic transmembrane segment and

Table 1. BibA protein conservation in GBS most common serotypes.

GBS strain Serotype Genea Surfaceb Secretedc

A909 Ia + – –515 Ia + – +DK1 Ia + +++ +5551 Ib/c + +++ +H36B Ib + ++ +2129 Ib + + +5518 Ib + – –18RS21 II + ++ +3050 II + +++ +5401 II + – +2141 II + ++ +M781 III + – +COH1 III + – –5435 III/R + – +5376 III/R + – –2274 IV + – +1999 IV + + +2603 V + +++ +CJB111 V + – +5364 V + ++ +2110 V/c + ++ +JM9130013 VIII + ++ +JMU071 VIII + ++ +1169 NT + ++ +

a. (+)bibA gene by PCR amplification.b. FACS analysis of BibA exposure on bacterial surface. –, MFI 0–50;+, MFI 50–100; ++, MFI 100–200; +++, MFI 200–300.c. Western blot analysis of BibA expression in the secreted proteinfraction. –, WB negative; +, WB positive.

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a charged C-terminal tail. Secondary structure predictionof BibA, carried out with the PHD program (Rost andSander, 1993), indicated strong helix propensity through-out the N-terminal domain and a high probability of coiled-coil arrangement for regions 283–294 and 366–400.

As shown in Fig. 2A, FACS analysis of 2603 V/R straingrown at exponential phase revealed a shift in bacterialfluorescence after staining with anti-BibA antibody. Thisindicated that BibA is exposed on the surface of GBS. Thisfinding was further confirmed by transmission immuno-electron microscopy (IEM), which showed positive immu-nogold labelling of BibA on the surface of 2603 V/R(Fig. 2B). Western blot analysis of 2603 V/R bacterialextracts showed the presence of a single band recog-nized by anti-BibA antibodies in both the mutanolysin-sensitive peptidoglycan protein fraction and culturesupernatant (Fig. 2C). Control experiments revealed thata cytosolic protein (SAG0274), predicted to be an alpha-glycerophosphate oxidase, was present in bacterialextracts but not in culture supernatants, excluding thepresence of contaminants from bacteria debris. The bandidentified as BibA has an apparent molecular weight (MW)of ~80 kDa, compared with the expected MW of 66 kDa.It is likely that the presence of a proline-rich motif inthe C-terminal region of BibA is responsible for thisdiscrepancy. Indeed, it is known that proline-rich regionsmay retard the electrophoretic migration of some proteins(Hollingshead et al., 1986). Comparative analysis of bac-

teria grown at exponential or stationary phases revealedno differences in the expression of BibA at the surface ofthe bacteria or secreted into the culture medium (data notshown). As expected, a mutant strain in which the bibAgene had been deleted showed neither BibA FACS posi-tive fluorescence nor immunogold surface labelling(Fig. 2D and E).

BibA is recognized by human Ig

Denaturing Western blotting analysis of recombinantBibA overlaid with purified human Ig showed that BibA isrecognized by both serum-derived IgG and serum andcolostrum-derived (secretory) IgA (Fig. 3A). Interestingly,purified mouse or bovine IgG did not recognize BibA(Fig. 3A). As expected, human IgG and IgA recognizedthe N-terminal portion of BibA, which is predicted to beexposed on bacterial surface. Human IgG also showedsome binding for the C-terminal portion, while thebinding of IgA was exclusively associated with theN-terminal portion of BibA (Fig. 3B). Surprisingly,Western blotting experiments showed no binding of BibAto purified Fc portion of human Ig, postulating the pres-ence of BibA-specific Ig. In order to assess the presenceof specific anti-BibA antibodies in human sera, we analy-sed five sera from normal healthy volunteers. By quan-titative ELISA we found that BibA-specific IgGconcentrations were in control sera in a range 0.9–

Fig. 1. Overview of sequence organization in BibA proteins. N-terminal domains (red) are predicted to form helix-rich structures, according topredictions obtained using the Paircoil program (Berger et al., 1995) at Expasy web server (http://www.expasy.org). C-terminal proline-richdomains (blue), classical (LPXTG) cell wall-anchoring motives (yellow) and trans-membrane domains (grey) are also indicated. Non-repetitiveinsertions (cyan) are characteristic of variants II and III.

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26.4 mg ml-1, with a geometric mean concentration of3.35 mg ml-1. The assay was highly specific, as demon-strated by the ability to inhibit the ELISA activity with theaddition of purified recombinant BibA protein as aninhibiting antigen (Fig. 3C). The addition of two un-related recombinant GBS surface proteins did not inhibitantibody binding to the BibA protein in this assay(Fig. 3C).

BibA binds to human complement regulator C4bp

Bacterial anti-phagocytic activity is often associated withthe ability to bind C4bp. As shown in Fig. 4, recombinantBibA, separated by SDS-PAGE (panel A) or spotted in thenative form onto nitrocellulose membrane (panel B),reacted strongly with C4bp overlaid at 5 mg ml-1. M proteinbound similarly to C4bp under both conditions, while a

negative control protein, randomly chosen from the 2603V/R genome (GBS201), did not bind. BibA N-terminal andC-terminal constructs were also tested for C4bp binding.Overlaid blots showed that the N-terminal region of BibAwas sufficient to specifically bind to C4bp (Fig. 4C). Nobinding was observed by the C-terminal portion. Interest-ingly, BibA did not show any binding for the alternativecomplement pathway regulator FH. Incubation of the2603 V/R strain with 125I radiolabelled C4bp revealed nobinding on bacterial surface, suggesting that the bindingsite for such a complement component is not easilyaccessible on whole bacteria.

BibA recombinant protein binds to epithelial cells

The in silico prediction of BibA propensity to form coiled-coil regions suggested an adhesive phenotype. We found

Fig. 2. BibA surface expression and release. Flow cytometry analysis of 2603 V/R strain (A), and 2603DbibA (D) incubated with a polyclonalmouse anti-BibA antibody and stained with PE-conjugated anti-mouse IgG antibody (black line histogram). The dashed line histogramindicates bacteria treated with primary and secondary antibodies alone. Immunogold electron microscopy of BibA expression in GBS strains2603 V/R (B) and 2603DbibA (E). Fixed bacteria were incubated with an anti-BibA serum and then labelled with secondary antibodyconjugated to 10 nm gold particles. Scale bars 200 nm. (C) Analysis of the presence of BibA in protein extracts from GBS strain 2603 V/R.Peptidoglycan-associated (p) and supernatant (s) protein fractions were separated by 10% (w/v) SDS-PAGE. Blots were overlaid with a mouseanti-BibA polyclonal antibody and stained with horseradish peroxidase-conjugated antibody.

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that a recombinant form of the 2603 V/R BibA binds toME180 cervical epithelial cells and that the bindingreached a plateau at a concentration of ~5 mg ml-1. Asshown in Fig. 5A, the binding of BibA to ME180 cells couldbe saturated. The affinity of recombinant BibA for its puta-tive receptor was therefore estimated by plotting the meanof fluorescence intensity of the BibA-receptor complexversus the free BibA concentration (Fig. 5B). The Kd valuewas then calculated as the BibA concentration that deter-mines the saturation of 50% of the putative receptorspresent on the cells, and was evaluated to be in the orderof ~4 ¥ 10-8 M. We also tested binding of recombinantBibA to intestinal (Caco2), pulmonary (A549) and bron-chial (16HBE) epithelial cell lines. Incubation of thesecells with 10 mg ml-1 of recombinant BibA significantlyincreased the mean of fluorescence of the BibA-receptorcomplex (Fig. 5C).

BibA is involved in GBS adhesion to epithelial cells

2603 V/R BibA deletion strain showed significantlyreduced binding to both ME180 and A549 cells (P < 0.05)(Fig. 6A). The impaired capacity of the 2603DbibA strainto adhere to epithelial cells was also evident in confocalimaging experiments. As shown in Fig. 6C, the numberof bacteria associated to epithelial cells was reduced inthe bibA mutant. These results were in complete agree-ment with those obtained by the association assay.Transformation of the 2603 V/R wild-type strain with thepAM401bibA plasmid was used as a tool to increase BibAexposure on bacterial surface. FACS experiments con-firmed that the 2603/pAM401bibA strain showed a signifi-cant increase in fluorescence intensity compared with thewild-type strain (data not shown). Association assaysdemonstrated that BibA overexpression was functionallyrelated to the capacity of GBS to adhere to epithelialcells. Indeed, compared with wild-type strain, 2603/pAM401bibA strain showed an increased adherence toboth ME180 and A549 cells (Fig. 6A).

Fig. 3. BibA is recognized by human Ig.A. Denaturing BibA immunoblotting overlaid with 0.5 mg ml-1 humanpurified serum IgG, human serum IgA, human secretory IgA,mouse serum IgG or bovine serum IgG. Each blot represents atypical experiment performed at least in triplicate.B. Full-length, N-terminal and C-terminal constructs of BibA wereseparated by SDS-PAGE and the blots overlaid with human IgG orhuman IgA.C. Results of competitive inhibition ELISA demonstrating antigenicspecificity of human antibodies reacting with plates coated withrecombinant BibA. Per cent inhibition of binding of human serum byeach inhibiting antigen (Ag) was determined by comparison ofabsorbance at 492 nm in the presence and absence of inhibitor.Black square labels indicate the mean � SD of the percentageinhibition by BibA of five human sera. White circle and white squarelabels indicate percentage inhibition by two unrelated GBS proteins.

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In the 515 Ia strain, which is predicted to carry a frame-shift within the LPXTG motif of bibA gene, BibA is exclu-sively found in the bacterial supernatant, but not in thepeptidoglycan-associated fraction (Fig. 6B). Introductionof pAM401bibA plasmid carrying the 2603 V/R form of BibAinto 515 Ia strain led to BibAtranslocation and anchoring onthe bacterial surface (Fig. 6B). In order to demonstrate thatsuch expression was associated to a functional adhesivephenotype, we compared association levels to epithelialcells between 515 Ia strain carrying the pAM401 plasmidand 515/pAM401bibA. We observed that BibAexposure on515 Ia surface resulted in a significant increase in thepercentage of associated bacteria to both ME180 andA549 cells (Fig. 6A). These findings were further confirmedby confocal microscopy imaging using the same experi-mental conditions as in association assay. As shown inFig. 6C, both 2603 V/R wild-type and BibA mutant strainswere uniformly distributed on epithelial cell surface and

clearly infection with the BibA mutant strain resulted in areduced number of bacteria associated to cells.

BibA knockout mutant strain is cleared more easily inhuman blood

The importance of BibA expression in bacterial survival invivo was assessed in freshly drawn blood from humandonors. As shown in Table 2, 2603 V/R wild-type andisogenic 2603DbibA knockout mutant strains were com-pared for the capacity to replicate in whole human blood.The bacterial survival index was calculated as the ratiobetween the number of bacteria recovered at the end ofthe assay versus bacteria at time zero. We tested fiveindividual donors and found that 2603 V/R wild-type strainproliferated in human blood five times more efficientlythan the 2603DbibA mutant strain. However, survivalindexes varied among the different donors. In three

Fig. 4. BibA binding to human C4bp.A. Immunoblotting of recombinant BibA overlaid with 5 mg ml-1 human C4bp. GAS M protein and GBS201 were used as positive and negativecontrols respectively.B. Dot blot of native recombinant BibA spotted onto nitrocellulose membrane at different concentrations (2.0–0.12 mg) and overlaid with5 mg ml-1 C4bp as in A.C. Western blotting of SDS-PAGE separated full-length, N-terminal and C-terminal constructs of BibA overlaid with human C4bp.

Table 2. BibA expression affects GBS survival in human whole blood.

Survival indexa (% survival relative to wild-type strain)

Donor A Donor B Donor C Donor D Donor E Mean � SD

2603 V/R 5.2 (100) 77.5 (100) 5.4 (100) 14.7 (100) 41.3 (100) 1002603 DbibA 0.7 (12.9) 37.6 (48.5) 0.76 (14.1) 1.3 (8.8) 5.4 (13.1) 19.5 � 14.6

a. Ratio between the number of bacteria recovered at the end of the assay versus bacteria at time zero.

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donors, where the wild-type strain replicated slowly inblood (from 5 to 14 times), the bibA knockout mutantstrain was almost cleared. By contrast, in two donorswhere the wild-type strain replicated highly in blood (77and 41 times), the bibA mutant strain still proliferated,although less efficiently (37 and 5 times respectively).These findings suggest that in donors with a reducedbactericidal activity, the contribution of BibA to GBS sur-vival in blood is less pronounced. Complementation of theBibA mutation by integration of the bibA gene and itsregulatory elements in the genome of the 2603DbibAstrain, restored roughly 60% of GBS survival phenotype inhuman blood.

BibA is involved in GBS anti-phagocytic activity

Phagocytic clearance of GBS by human blood is mainlymediated by polymorphonuclear leucocytes (PMNs),which kill opsonized bacteria in the presence of comple-ment (Edwards et al., 1985; Cheng et al., 2001). Thekilling of the 2603 V/R wild-type and the bibA mutantstrains by human PMNs were compared. Mid-exponential phase bacteria opsonized with normalhuman serum were incubated with PMNs and thenumber of surviving bacteria was determined by quanti-tative plating on TSA plates. As shown in Fig. 7 the bibAmutant was killed more efficiently than the wild-type

Fig. 5. Binding of recombinant BibA to epithelial cells.A. ME180 cells were incubated for 1 h at 4°C with increasing concentrations of recombinant BibA (range 0.01–62.5 mg ml-1). Cells wereincubated with mouse anti-BibA antibodies followed by PE-conjugated secondary antibodies and analysed by flow cytometry. MFI, meanfluorescence intensity.B. Saturation curve of BibA binding to ME180 cells. Analysis was performed on data reported in A. The Kd value was calculated as the BibAconcentration that determines the saturation of 50% of the receptors present on the cells.C. Representative flow cytometry profiles of the binding of 10 mg ml-1 BibA to A549, Caco2 and 16HBE epithelial cells. Dashed line histogramsrepresent the binding to control cells incubated with the anti-BibA antiserum followed by fluorescent conjugated secondary antibodies.

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strain. Indeed, after 3 h post infection only 21% of themutant bacteria survived phagocytosis by PMNs, com-pared with 52% of the wild-type strain. In control experi-ments, the initial colony-forming units (cfu) count did notvary significantly when opsonized bacteria were incu-bated for 3 h at 37°C without PMNs.

BibA contributes to the virulence of GBS in the mouse

We studied the role of BibA in the virulence of GBS byinfecting mice with the wild-type strain and the bibAmutant. Over a period of 10 days, we monitored the mor-tality of mice infected intraperitoneally (i.p.) with a range of

Fig. 6. BibA expression modulates GBS capacity to adhere to epithelial cells.A. ME180 cells and A549 cells grown in a 24 well plate were infected with GBS strains 2603 V/R, 2603DbibA, 2603/pAM401bibA, 515/pAM401and 515/pAM401bibA for 2 h. Bacterial association was calculated in relation to the wild-type strain (100%). Data represent the meanvalues � standard deviations of three individual experiments. P < 0.05.B. Analysis of the presence of BibA on bacterial surface (IEM) and protein extracts (PAGE) from GBS strains 515/pAM401 and515/pAM401bibA.C. Confocal imaging analysis of the 2603 V/R strain association to A549 lung epithelial cells in comparison with the isogenic DbibA mutantstrain. 515/pAM401 wild-type strain was analysed in comparison with 515/pAM401bibA. A549 cells grown on glass slides were infected withGBS for 2 h. Bacteria were stained with mouse polyclonal anti-capsular antibodies (red) and BibA with rabbit polyclonal anti-BibA antibodies(green). Magnification, ¥20.

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doses from 5 ¥ 107 to 5 ¥ 103 bacteria. As shown in Fig. 8,at a dose of 5 ¥ 105 bacteria 50% of the mice infected with2603 V/R wild-type strain were dead within 3 days, whileonly 10% of mice infected with bibA mutant strain weredead by the same period. No further killing was observedfor both strains up to 10 days post infection. Infection withhigher doses of bacteria (5 ¥ 107 and 5 ¥ 106) resulted in100% killing of mice by both wild-type and mutant strainswithin 1 day, while at lower dose all mice survived.

Discussion

BibA is a widely expressed protein and in silico analysis ofthe seven GBS completed genomes revealed that BibA isa modular protein, whose sequence variability is mainlydue to a different number of short amino acid repeats,either in the N-terminal or in the C-terminal domains. ThebibA gene is located between secE and nusG genes,which are co-transcribed in Escherichia coli (Downinget al., 1990) and have been found to be adjacent in a largepanel of Gram positive and Gram negative bacteria(Jeong et al., 1993; Miyake et al., 1994; Sharp, 1994;Puttikhunt et al., 1995; Katayama et al., 1996; Syvanenet al., 1996; Fuller et al., 1999; Poplawski et al., 2000;Barreiro et al., 2001). This suggests that the presentgenomic localization of bibA is likely to derive from aninsertion event.

Of particular interest, two transposases present in A909strain are members of the IS1381 element, recently pro-posed as a tool for GBS and S. pneumoniae subtyping(Tamura et al., 2000). In particular, the presence ofIS1381 has been correlated with the evolution of the

Streptococcus agalactiae species analysed by multilocussequence typing (Hery-Arnaud et al., 2005). A generalfeature of IS elements is that upon insertion, most gener-ate short directly repeated sequences of the target DNA.Sequence analysis of IS1381 from A909 strain suggeststhat the nucleotide region coding IKAESIN is the directrepeat originated by IS1381 insertion. Although the ISelement is exclusively found within A909 allele, multiplecopies of the IKAESIN motif were identified in CJB111 andH36B bibA variants. This suggests that multiple insertion/excision events might have regulated bibA gene expres-sion during GBS evolution. A BLAST search against thenon-redundant GenBank database did not reveal signifi-cant homologies of BibA with other bacterial proteins,apart from a weak similarity in the N-terminal region witha series of streptococcal Ig-binding proteins such asthe M protein family (22% identity) of GAS (Podbielskiet al., 1994), PspC or Hic (20% identity) of S.pneumoniae (Dave et al., 2004) and Mig (27% identity) ofS. dysagalactiae (Song et al., 2002).

BibA is structurally related to the family of M-like pro-teins of GAS. Indeed, it is known that the secondarystructure of M proteins is primarily an a-helical coiled-coilstructure forming stable dimers (Phillips et al., 1981).In silico prediction of BibA secondary structure (Bergeret al., 1995) revealed a helix-rich region within theN-terminal portion with the propensity to form a coiled-coilarrangement. No canonical elements of secondary struc-ture are, however, predicted within the proline-rich region,suggesting that this part of the molecule could adopt apoly-proline helix-like conformation.

BibA, when either expressed on the cell wall orsecreted in the supernatant, seem to have an identicalMW. This suggests that secretion of BibA might be due to

Fig. 7. BibA promotes GBS survival of PMN killing. Humanneutrophils were incubated for 1, 2 and 3 h with GBS 2603 V/R(black circle) and 2603DbibA mutant strain (black triangle) in thepresence of normal human serum. Percentage of viable bacteriaafter incubation with neutrophils is reported. A typical experimentperformed in triplicate is shown.

Fig. 8. BibA expression increases the virulence of GBS in mice.Mortality curves in mice infected with GBS 2603 V/R strain (blacksquare) and the isogenic bibA mutant strain (black circle). Mice(10 per group) were inoculated i.p. with 5 ¥ 105 bacteria.

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a proteolytic cleavage of the cell wall-anchoring domain. Arecently identified Ig-binding protein from GAS (SibA) hasalso been reported in both whole-cell protein fractions andculture supernatants after overnight growth (Fagan et al.,2001).

Of interest, BibA has a YSIRK-G/S-like motif in thesignal peptide, which has been postulated to be involvedin accelerating the maturation of proteins in Staphylococ-cus aureus and other Gram positive pathogens (Bae andSchneewind, 2003).

In order to survive phagocytic killing, GBS has devel-oped a series of strategies that selectively interfere withthe complement alternative pathway (Marques et al.,1992), oxidative burst (Liu et al., 2004) and antimicrobialpeptides (Poyart et al., 2003). In particular, GBS sialicacid-rich capsule prevents C3b deposition (Wessels et al.,1989; Marques et al., 1992). The binding of purified BibAto C4bp has led us to hypothesize a role for this protein inGBS anti-phagocytic activity. Indeed, it has been shownthat the ability of GAS to selectively bind C4bp fromhuman serum correlates with its capacity to evade phago-cytosis (Berggard et al., 2001). However, when we testedthe in situ binding of C4bp to both 2603 V/R wild-typestrain and the bibA isogenic mutant strain, no binding wasobserved. This is not completely unexpected, as it hasbeen previously shown that among 17 GBS strains testednone of them had the capacity to bind C4bp on bacterialsurface (Thern et al., 1995). However, as BibA is alsofound in GBS bacterial culture supernatants, we cannotexclude that the protein has a functional binding C4bp insuch conformational conditions. Indeed, our findings thatGBS survives better in human whole blood when express-ing BibA on its surface and that opsonized GBS lackingBibA is cleared more efficiently in a complement-mediatedopsonophagocytic assay, support the hypothesis of an invivo anti-phagocytic role of the protein. This is furthercorroborated by the fact that in a mouse infection model,after i.p. injection the 2603 V/R strain is more virulent thanthe isogenic bibA mutant strain.

Bacterial adherence to host cells is the initial step and aprerequisite for successful colonization of host mucosalsurfaces. Analysis of the binding of recombinant BibA toME180 epithelial cell line revealed that its associationcould be saturated, with an estimated affinity constant of~4 ¥ 10-8 M. Furthermore, BibA binding to epithelial celllines of pulmonary, intestinal, bronchial and cervical originsuggests the existence of an ubiquitous receptor. Thus,like M proteins, BibA mediates bacterial adhesion to epi-thelial cells (Courtney et al., 1994; 1997; Wang andStinson, 1994). Expression of the cell-wall anchored formof BibA in a strain not exposing BibA on its surface,increased its binding capabilities in both human cervical(ME180) and lung (A549) epithelial cell lines, which aretargets for GBS colonization.

The immunogenicity of the protein led us to hypothesizethat anti-BibA antibodies might protect humans from GBSinfections. However, immunization of mice with recombi-nant BibA did not induce protective antibodies (D. Maioneand M. Soriani, unpubl. results). Further studies need tobe carried out in order to elucidate the functional outcomeof BibA-specific Ig produced in response to GBSinfections. In conclusion, we believe that the discovery ofthis potential new virulence factor, involved both in GBSinteractions with host cells and in resistance to phagocytickilling, represents an important achievement in combatingthe life-threatening infective disease associated to thisbacterium.

Experimental procedures

Bacterial strains and growth conditions

Group B Streptococcus strains 2603 (serotype V/R), 515(serotype Ia) and were used in this study. In order to deter-mine BibA protein conservation, we analysed a panel of GBSstrains, kindly provided by G. Orefici (ISS, Rome, Italy).E. coli DH5a and DH10BT1 were used for cloning purposesand E. coli BL21 (DE3) for expression of BibA fusion protein.GBS was cultured at 37°C in Todd–Hewitt broth (THB) up toOD600 0.4. GBS strains carrying the plasmid pAM401bibAwere grown in the presence of chloramphenicol (10 mg ml-1).E. coli was grown in Luria–Bertani broth; E. coli clones car-rying the plasmids pAM401bibA, pJRS233DbibA or pET21(b)+ derivatives were grown in the presence of chloramphenicol(20 mg ml-1), erythromycin (400 mg ml-1) or ampicillin(100 mg ml-1) respectively.

Sequence analysis

The alignment of 2603 V/R (GenBank Accession NumberNP_689049), 18RS21 (AAJO00000000), 515 Ia(AAJP00000000), NEM316 (NP_736451), H36B(AAJS00000000), CJB111 (AAJQ00000000), A909(YP_330593) and COH1 (AAJR00000000) strains was per-formed using CLUSTALW (Thompson et al., 1994).

Construction of 2603 V/R bibA deletion mutant andcomplemented strain

The bibA gene was deleted in GBS 2603 V/R strain accordingto the procedure previously described (Lauer et al., 2005).The in frame deletion fragment was obtained by SplicingOverlap Extension PCR using the primers P1, P2, P3 and P4.The XhoI-digested fragment was cloned into the XhoI-digested pJRS233 plasmid. After cloning the in frame dele-tion fragment in pJRS233, the plasmid pJRS233DbibA wasobtained.

The plasmid pJRS233DbibA was then transformed into the2603 V/R strain by electroporation and transformants wereselected after growth at 30°C on agar plates containing1 mg ml-1 erythromycin. Transformants were then grown at37°C with erythromycin selection as previously described

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© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 754–767

(Maguin et al., 1996). Integrant strains were serially pas-saged for 5 days in liquid medium at 30°C without erythro-mycin selection to facilitate the excision of plasmidpJRS233DbibA, resulting in the bibA deletion on thechromosome. Dilutions of the serially passaged cultures wereplated onto agar plates, and single colonies were testedfor erythromycin sensitivity to confirm the excision ofpJRS233DbibA.

For the complemented strain, the bibA gene was integratedinto the pseudogene SAG1543. This pseudogene waschosen for the presence of a unique NdeI restriction site. Theintegration region including the pseudogene SAG1543 plusthe flanking regions was amplified using the primers C1 andC2. The PCR products were cloned into the BamHI/SalI-digested pAM401 vector, resulting in plasmid pIS1. The bibAgene including its own promoter and terminator was amplifiedby PCR from chromosomal DNA of 2603 V/R strain usingprimers C3 and C4. The NdeI-digested PCR product wascloned into the NdeI-digested plasmid pIS1. This construct,named pIS2, was digested with XhoI, incorporated in the 5′ends of primer C1 e C2, to release the fragment containingthe bibA gene plus the flanking regions. The resulted XhoI-digested fragment was cloned into the pJRS233 plasmidresulting in pIS3 that was used to transform 2603DbibA strainby electroporation. The complemented strains were selectedas previously described for the construction of bibA knockoutstrain. The expression of the protein BibA in the comple-mented strain was confirmed by Western blot on bacterialsurface protein fraction and supernatant and by FACS.Primers details are reported on Table 3.

Plasmid-mediated expression of BibA in GBS

The bibA gene including its own promoter and terminator wasamplified by PCR from chromosomal DNA of GBS 2603 V/R

using primers O1 and O2. The BamHI/SalI-digested PCRproducts were cloned into the BamHI/SalI digested E. coli–S. faecalis pAM401 shuttle vector. The plasmid pAM401bibAwas obtained by cloning the bibA gene into pAM401. PlasmidpAM401bibA was transformed by electroporation into 2603V/R and 515 Ia strains with subsequent chloramphenicolselection.

Fluorescence-activated cell sorter analysis

In order to quantify the exposure of BibA on the bacterialsurface, GBS was grown up to OD600 0.4 and incubated withrabbit anti-BibA serum. Bacteria were then washed and incu-bated with the R-Phycoerytrin (PE)-conjugated secondaryantibodies (Jackson Immuno Research, PA, USA) at 4°C.After washing, bacteria were fixed with 2% PFA for 20 min atroom temperature (RT) and analysed by a FACSscan flowcytometer (Becton Dickinson). In binding assays, epithelialcells were mixed with different concentrations of BibA andincubated for 1 h at 4°C. Cells were subsequently incubatedfor 45 min at 4°C with rabbit anti-BibA serum in 5% FCS andbinding revealed by PE-conjugated secondary antibodies.Cell-bound fluorescence was analysed by FACS.

Cell culture

The human cervical epithelial cell line, ME180, was pur-chased from the American Type Culture Collection (Rockville,MD). Cells were maintained in RPMI 1640 medium with 10%heat-inactivated fetal bovine serum (FBS). The human coloncarcinoma epithelial cell line Caco2 and the human lungcarcinoma epithelial cell line A549 were supplied by the ATCCand grown in DMEM supplemented with 10%v FBS, 4.5 g l-1

glucose and non-essential amino acids.

Table 3. List of primers used in this study.

Primer Sequence (5′ to 3′) Restriction site

Recombinant proteinsBibA F5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ NdeI

R5′-CCCGCTCGAGAATTGCTAAGAGTGGACTTGC-3′ XhoIBibA-N term (34–394) F5′-GGAATTCCATATGCACGCGGATACTAGTTCAGGA-3′ NdeI

R5′-CCCGCTCGAGACCTCTGGTAAGGTCTTGAA-3′ XhoIBibA-C term (389–622) F5′-GGAATTCCATATGCCAGACCTTACCAGAGGT-3′ NdeI

R5′-CCCGCTCGAGCGTAATAAGACCTGCACTT-3′ XhoI2603DbibA

P1 F5′-CCCGCTCGAGACTAGTGACAAACCTTGGAAT-3′ XhoIP2 R5′-GTCAGCACGGTTTGCCATAAACCGAAAGGTCTATCC-3′P3 F5′-ACCTTTCGGTTTATGGCAAACGCTGCTGACATTG-3′P4 R5′-CCCGCTCGAGACAGATAAGCCTAAGCGACTT-3′ XhoI

2603/pAM401bibA 515/pAM401bibAO1 F5′-CCCCGCCGGGATCCCCAACCCTTATCAAAAGA-3′ BamHIO2 R5′-CTCTGCATGGTCGACATAGAAACAACCCAAACCC-3′ SalI

2603DbibA::bibAC1 F5′-CCGCCGGGATCCctcgagTTGAGCAGCACGCTTAAA-3′a BamHIC2 R5′-CTCTGCATGGTCGACctcgagGGGGCGCTGAAAATTATG-3′a SalIC3 F5′-GGAATTCCATATGCCAACCCTTATCAAAAGA-3′ NdeIC4 R5′-GGAATTCCATATGATAGAAACAACCCAAACC-3′ NdeI

a. In bold XhoI restriction site.F correspond to forward primer and R to reverse primer. Restriction sites are underlined.

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Adhesion assay

Human epithelial cells were infected with GBS in infectionmedium (basal medium without antibiotics) supplementedwith 2% FBS in 200 ml volumes. After 2 h incubation at 37°Cin 5% CO2 (v/v), total cfu were estimated after addition of 1%saponin to the wells contents. Adhesiveness was quantifiedby determining the ratio of cell-associated cfu versus total cfupresent in the assay.

Immunogold labelling and electron microscopy

Overnight cultures of GBS strains were grown in THB at 37°Cup to OD600 0.3. Formvar-carbon-coated nickel grids werefloated on drops of GBS suspensions. The grids were thenfixed in 2% PFA for 5 min, and placed in blocking solution for30 min. The grids were then incubated with primary antise-rum against BibA diluted 1:20 in blocking solution for 30 minat RT, washed with blocking solution, and floated on second-ary antibody conjugated to 10 nm gold particles for 30 min.The grids were examined using a TEM GEOL 1200EX IItransmission electron microscope.

Confocal immunofluorescence microscopy

Epithelial cells grown on polycarbonate (PET) filters wereinfected with bacteria at a multiplicity of infection of 10:1 andincubated at 37°C for 2 h. Cells were washed three times inPBS and fixed in 2% paraformaldehyde for 30 min at RT or at4°C overnight. After fixing, the PET membrane supporting thecells was cut and inserted into a multiwell plate, where therest of the protocol was performed. Cells were then perme-abilized by immersion in 0.1% Triton X-100 in PBS containing0.05% Tween20 (PBS-T) for 20 min at RT. After a secondPBS-T wash the cells were then stained with phalloidin con-jugated with Alexa Fluor dye A620 (Molecular Probes) inPBS-T for 30 min at RT. Phalloidin was then washed offwith PBS-T and the cells incubated for 30 min with 3% BSA inPBS-T. The monolayers were then incubated with a mix ofmouse anti-capsule and rabbit anti-BibA polyclonal antibod-ies for 1 h at RT. Bacteria were then stained with goat anti-mouse and anti-rabbit Alexa Fluor (Molecular Probes)conjugated antibodies (excitation at 568 nm and 488 nmrespectively). Slow Fade reagent kit (Molecular Probes) wasused to mount coverslips. The slides were viewed with aBio-Rad confocal scanning microscope.

Human whole blood killing assay

Group B Streptococcus was grown up to OD600 0.4, washedand resuspended in PBS. A total of 104 cfu in 100 ml weremixed with 300 ml of freshly drawn human blood usingheparin as anticoagulant. The tubes were incubated for 3 hwith agitation at 37°C and dilutions were plated for determi-nation of cfu.

Killing of GBS by human neutrophils

Polymorphonuclear leucocytes were obtained from heparin-anticoagulated venous blood of normal healthy volunteers aspreviously described (Maione et al., 2005). GBS was grownup to mid-log phase and 106 cfu were opsonized for 15 min at

37°C with 5% normal human serum. Pre-warmed neutrophils(106) were then added to opsonized GBS and incubated at37°C on a thermomixer up to 3 h. PMNs/bacteria suspen-sions were diluted in ice-cold distilled water to disrupt PMNsand serial dilutions were plated onto TSA plates to determinethe number of viable bacteria.

Mouse virulence assay

Pathogen-free CD1 female mice (6 weeks old) were used inthis study. Groups of 10 mice were inoculated i.p. with dosesranging between 5 ¥ 107 and 5 ¥ 103 cfu of 2603 V/R wild-type strain and 2603DbibA strain. The mortality was observedover a 10 day period.

ELISA measurement of BibA-specific antibodies inhuman serum

BibA-specific antibodies were quantified by ELISA on humansera. Serial twofold dilutions of sera were dispensed inmedium binding 96 well plates (Greiner Bio-One) coatedovernight with recombinant BibA at 1 mg ml-1 in PBS. Anti-body binding was revealed by horseradish peroxidase-conjugated anti-human IgG, followed by the substrate OPD(Sigma). Levels of BibA-specific IgG in serum samplesobtained from five normal healthy volunteers were measuredto compare optical density measurements from the BibA-specific IgG ELISA to a standard curve. The standard curvewas generated by an ELISA that used unlabelled goat anti-human IgG to coat plates and serial twofold dilutions of unla-belled affinity-purified human IgG starting at 0.1 mg ml-1.

Inhibition ELISA was performed adding to plates coatedwith recombinant BibA (1 mg ml-1) a previously incubatedmixture of normal healthy volunteers serum samples at theserum dilution giving an A492 between 1.6 and 1.8 withincreasing concentrations (from 0.0001 to 100 mg ml-1) of thefollowing inhibiting antigens: purified recombinant BibAprotein or purified recombinant unrelated GBS proteinswhose MW was approximately identical to BibA. Percentinhibition was calculated as follows: [(A492 of uninhibitedcontrol - A492 of test sample)/(A492 of uninhibitedcontrol)] ¥ 100.

The following methodologies have been included inSupplementary material: BibA recombinant protein expres-sion and purification, bacterial extracts and Dot blot andWestern blot analysis.

Acknowledgements

The authors have no conflicting financial interests. This workwas supported by the company internal funding. We thankVittoria Pinto for technical assistance in protein purification.We are indebted to Enrico Luzzi for the useful advices onthe ELISA methodology and human serology. We thankDomenico Maione and Daniela Rinaudo for the useful dis-cussion on anti-phagocytic mechanisms. We thank GiulianoBensi for providing the recombinant M protein of GAS. Weare indebted to Laura Serino for sharing ideas andsuggestions. We also thank Rino Rappuoli, Robert Janulc-zyk, Andrew Edwards and Angela Nobbs for critical reading ofthe manuscript.

BibA: a novel multifunctional protein of GBS 765

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 754–767

Note added in proof

Graphic representation of BibA sequence organization shownin Fig. 1 confirms the allelic variability of the protein, asrecently reported by Lamy et al. [Lamy, M.C., Dramsi, S.,Billoet, A., Reglier-Poupet, H., Tazi, A., Raymond, J., et al.(2006) Rapid detection of the ‘highly virulent’ group B Strep-tococcus ST-17 clone. Microbes Infect 8: 1714–1722.]

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Supplementary Material

The following supplementary material is available for thisarticle online:Appendix S1. BibA recombinant protein expression andpurification, bacterial extracts and Dot blot and Western blotanalysis.

This material is available as part of the online article fromhttp://www.blackwell-synergy.com

BibA: a novel multifunctional protein of GBS 767

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 754–767