Assembly and role of pili in group B streptococci

Molecular Microbiology (2006) doi:10.1111/j.1365-2958.2006.05190.x

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology0950-382X© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd

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Original Article

Pili of Streptococcus agalactiaeS. Dramsi

et al.

Accepted 3 March, 2006. *For correspondence. E-mail [email protected]; Tel. (

+

33) 1 44 38 94 87; Fax (

+

33) 1 45 68 89 38.

Assembly and role of pili in group B streptococci

Shaynoor Dramsi,

1

* Elise Caliot,

1

Isabelle Bonne,

2

Stéphanie Guadagnini,

2

Marie-Christine Prévost,

2

Mila Kojadinovic,

1

Lila Lalioui,

1

Claire Poyart

1,3

and Patrick Trieu-Cuot

1

1

Unité de Biologie des Bactéries Pathogènes à Gram-Positif Institut Pasteur, URA CNRS 2172, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.

2

Plate-forme de microscopie électronique, Institut Pasteur Paris, France.

3

INSERM 567, Institut Cochin, Département de Maladies Infectieuses, Faculté de Médecine René Descartes, Université Paris 5, 24 rue du faubourg Saint Jacques, 75014 Paris, France.

Summary

Streptococcus agalactiae

[group B streptococcus(GBS)] is the leading cause of neonatal pneumonia,sepsis and meningitis. An

in silico

genome analysisindicated that GBS strain NEM316 encodes five puta-tive sortases, including the major class A sortaseenzyme and four class C sortases. The genes encod-ing the class C sortases are tandemly arranged intwo different loci,

srtC1-C2

and

srtC3-C4

, with a sim-ilar genetic organization and are thought to beinvolved in pilus biosynthesis. Each pair of sortasegenes is flanked by LPXTG protein encoding genes,two upstream and one downstream, and a diver-gently transcribed regulatory gene located upstreamfrom this locus. We demonstrated that strain NEM316expresses only the

srtC3-C4

locus, which encodesthree surface proteins (Gbs1474, Gbs1477 andGbs1478) that polymerize to form appendagesresembling pili. Structural and functional analysis ofthis locus revealed that: (i) the transcriptional activa-tor RogB is required for expression of the

srtC3-C4

operon; (ii) Gbs1477, and either SrtC3 or SrtC4 areabsolutely required for pilus biogenesis; and (iii)GBS NEM316 pili are composed of three surface pro-teins, Gbs1477, the

bona fide

pilin which is the majorcomponent, Gbs1474, a minor associated compo-nent, and Gbs1478, a pilus-associated adhesin. Sur-prisingly, pilus-like structures can be formed in theabsence of the two minor components, i.e. the puta-

tive anchor Gbs1474 or the adhesin Gbs1478. Adher-ence assays showed that Gbs1478 confers adhesivecapacity to the pilus. This study provides the firstevidence that adhesive pili are also present in Gram-positive pathogens.

Introduction

Streptococcus agalactiae

[also known as group B strep-tococcus (GBS)] is commonly found in humans, where itis present in the gastrointestinal tract and vagina (20–30%) of healthy individuals. However, in certain circum-stances, GBS can become a life-threatening pathogen,causing invasive infections in human neonates. Thus,adaptation to changing environments and adhesion tovarious host surfaces are key steps in GBS pathogenesis.Surface proteins play important roles during variousstages of GBS infection as they contribute to adhesion toepithelial cells, interactions with the human extracellularmatrix and escape from host immunity (Lindahl

et al

.,2005). Binding of GBS to various extracellular matrix com-ponents has been characterized at the molecular level.Interaction with human laminin is mediated by the lipopro-tein Lmb (Spellerberg

et al

., 1999). Interaction with humanfibrinogen is mediated by the cell wall-anchored proteinFbsA (Schubert

et al

., 2002) and to a lesser extent by asecreted protein FbsB (Gutekunst

et al

., 2004). Binding toimmobilized fibronectin is mediated by the cell wall-anchored protein ScpB (Beckmann

et al

., 2002; Cheng

et al

., 2002).Gram-positive bacteria have evolved a variety of differ-

ent mechanisms to display proteins on the cell surface(reviewed by Navarre and Schneewind, 1999; Cossartand Jonquieres, 2000), one of which, referred to as ‘sort-ing’, results in the covalent attachment of the protein topeptidoglycan. Cell wall-anchored surface proteins aremade with a signal peptide at their amino-terminus and acharacteristic carboxy-terminal sorting signal: a con-served LPXTG motif followed by a hydrophobic domainand a positively charged tail (Fischetti

et al

., 1990). Fol-lowing secretion, the sorting signal is cleaved between thethreonyl and glycyl residues of the LPXTG motif and thecarboxyl group of the threonine is amide-linked to theamino group of cross-bridges within peptidoglycan precur-sors (Ton-That

et al

., 2000). The enzyme that catalysesthe protease and transpeptidase activities is a membrane-associated protein named sortase (Srt) (Mazmanian

et al

., 1999).

2

S. Dramsi

et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Molecular Microbiology

Sequence analysis of the three complete GBSgenomes, serotype III strain NEM316 (Glaser

et al

.,2002), serotype V strain 2603V/R (Tettelin

et al

., 2002)and serotype Ia strain A909 (Tettelin

et al

., 2005),revealed the presence of 35, 23 and 19 genes encodingLPXTG-containing proteins respectively, and five genesencoding sortases. Genome analysis of sortases inGram-positive bacteria led to the identification of four [A,B, C and D (Dramsi

et al

., 2005)] or five [A and B, families3, 4 and 5 (Comfort and Clubb, 2004)] structural classesdepending upon the approach utilized. The class Aenzymes, the prototype of which is the sortase A (SrtA)of

Staphylococcus aureus

, likely acts on most if not allLPXTG-containing proteins. The SrtA contain a hydropho-bic NH2-terminal segment functioning both as a signalsequence and as a membrane anchor sequence, and acarboxylic catalytic signature sequence (TLXTC) contain-ing an essential cysteyl residue (Ilangovan

et al

., 2001;Ton-That

et al

., 2002). Genes encoding SrtA are ubiqui-tous among low GC% Gram-positive bacteria except forsome strains of

Streptococcus thermophilus

, whereasthose encoding class B, C or D are restricted to a limitednumber of sequenced genomes (Dramsi

et al

., 2005).Class B and C sortases were shown to be required foranchoring a small number of substrates (Barnett andScott, 2002; Mazmanian

et al

., 2002).We previously demonstrated that a GBS SrtA

–

mutantwas impaired for binding to the major extracellular matrixcomponents (fibronectin and fibrinogen) and for adher-ence to various human epithelial cells (Lalioui

et al

.,2005). Besides

srtA

, four genes encoding class C sor-tases (SrtC) were found in NEM316, 2603V/R and A909genome sequences. Class C sortases have been impli-cated in the polymerization of LPXTG-containing pilin sub-units in actinomycetes (Ton-That and Schneewind, 2003;Ton-That

et al

., 2004) and, more recently in group Astreptococci (Mora

et al

., 2005). While this work wasin progress, two LPXTG proteins encoded by a SrtC-encoding loci in GBS strain JM9130013 of serotype VIIIwere shown to be present in pilus-like structures (Lauer

et al

., 2005). However, the mechanism of assembly andthe functional role of these pili are yet unknown.

In this work, we carried out a detailed structural andfunctional analysis of the SrtC-encoding loci of GBSNEM316. We report that, in this strain, pilus biosynthesisis due to expression of only one of its two

srtC

loci andthat this appears to be a common feature of GBS strains.

Results

In silico

characterization of the sortases loci in GBS

Sortases are involved in the cell wall anchoring of proteinscontaining a conserved carboxylic sorting motif known as

the LPXTG motif. We compared the structure of the threeloci encoding sortases in GBS strains NEM316, 2603V/Rand A909. As shown in Fig. 1A, the structure of the

srtA

locus is identical in the three strains, and the role of thecorresponding enzyme was previously characterized instrain NEM316 (Lalioui

et al

., 2005).In GBS, the four other sortases were members of the

class C structural group according to our recent classifi-cation (Dramsi

et al

., 2005). They were found tandemlyarranged in two different loci possessing a similar geneticorganization (Fig. 1). Each pair of sortase genes isflanked by two upstream and one downstream genesencoding LPXTG proteins, and this putative operon islikely regulated by an upstream, divergently transcribedregulatory gene. The first locus encoding the sortases C,

srtC gbs0630

and

srtC gbs0631

, hereafter designated

srtC1

and

srtC2

, is highly conserved in the three GBSsequenced strains (99.9% at the DNA level). At least oneLPXTG protein encoded by this locus contains the canon-ical pilin-like motif (I/VYPKN) as defined by Ton-That andSchneewind (2003). Interestingly, in NEM316, the geneencoding the putative transcriptional regulator of the AraCfamily (

sag0644

in strain 2603V/R) was interrupted byan in-frame stop codon to give

gbs0627

and

gbs0626

(Fig. 1B). The second SrtC-encoding locus,

srtC gbs1475

and

srtC gbs1476

, hereafter designated

srtC3

and

srtC4

,is less conserved in GBS strains (Fig. 1C).

In NEM316 and 2603V/R, the

srtC1-C2

and

srtC3-C4

loci are similar. They both contain six genes encoding atranscriptional regulator, three LPXTG proteins, and twoSrtC (Fig. 1B and C). Of note, in NEM316, the Gbs1478protein is a paralogue of Gbs0632 (52.4% similarity),Gbs1477 is related to Gbs0628 (29% similarity), andGbs1474 to Gbs0629 (38% similarity). Comparison of the

srtC3-C4

locus in NEM316 and 2603V/R revealed tworemarkable features: (i) the inactivation of the transcrip-tional regulator RogB in 2603V/R by a frameshift mutation,mirroring that found in the AraC-like regulator of

srtC1-C2

locus in NEM316 (Fig. 1B and C) and (ii) a gene replace-ment,

gbs1477

to

sag1407

, encoding orthologous pro-teins, but only displaying 61% similarity (Fig. 1C). In strainA909, the

srtC3-C4

locus, as previously defined, isreplaced by a segment encoding two LPXTG proteinsdevoid of a pilin motif that are associated with two com-plete (Sak1439 and Sak1437) and one truncated SrtC(Sak1438) (Fig. 1C).

BLAST

searches with genes encoding the LPXTG-containing paralogues Gbs0628/Gbs1477, Gbs0629/Gbs1474 and Gbs0632/Gbs1478 as queries revealed astriking degree of conservation in gene order, composi-tion, and organization with the pilus locus described in

Corynebacterium diphteriae

(Ton-That and Schneewind,2003). From the pilus assembly model proposed in

C. diphteriae

, Gbs0628/Gbs1477 might be the major pilin

Pili of

Streptococcus agalactiae 3

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Molecular Microbiology

subunit because of the presence of a canonical pilin motif,Gbs0629/Gbs1474 and Gbs0632/Gbs1478, minor pilus-associated subunits.

Electron microscopy evidence for pilus-like structure

To determine whether SrtC loci code for pilus-like struc-tures, antibodies raised against recombinant putative pilinproteins (i.e. Gbs0628 and Gbs1477) were used toexplore the protein localization by immunogold electronmicroscopy (IEM) in strain NEM316. Labelling was notobserved with antibodies directed against Gbs0628 (datanot shown). In contrast, IEM with an antiserum specific for

Gbs1477 revealed abundant surface staining and longpilus-like structures extending up to 500 nm from the bac-terial surface (Figs 2 and 3, WT strain). These results areconsistent with those of a transcriptome analysis revealingthat, in NEM316, the

srtC1-C2

locus is expressed at a lowlevel, whereas the

srtC3-C4

locus is expressed at a highlevel (P. Glaser, pers. comm.). In addition to

gbs1477

gene, the

srtC3-C4

locus contains two other genes(

gbs1478

and

gbs1474

) predicted to encode cell wall-anchored proteins (Fig. 1). Specific antiserum raisedagainst Gbs1474 revealed surface staining with anuneven distribution of the gold particles, but pili were notobserved (Figs 2 and 3).

Fig. 1.

Structure of the sortases encoding loci,

srtA

(A) and

srtC

(B and C), in three sequenced GBS strains: serotype III strain NEM316 (Glaser

et al

., 2002), serotype V strain 2603V/R (Tettelin

et al

., 2002), and serotype Ia strain A909 (Tettelin

et al

., 2005).A. The

srtA

locus which encodes the major sortase designated SrtA is identical in NEM316, 2603V/R and A909. As in all streptococcal genomes sequenced so far, the

srtA

gene is located downstream from the

gyrA

gene.B. The

srtC1-C2

locus is identical in strains NEM316, 2603V/R and A909 except for one mutation in NEM316 that introduces an in-frame stop codon in the regulatory gene

sag0644

. This locus contains two C sortases genes

gbs0630

and

gbs0631

, thereafter designated

srtC1

and

srtC2

respectively, which are flanked by genes coding for three LPXTG-containing proteins (

gbs0628

,

gbs0629

and

gbs0632

). The genes

gbs0633

and

gbs0634

encode truncated sortases.C. The

srtC3-C4

locus is almost identical in strains NEM316 and 2603V/R. It comprises one regulatory gene

gbs1479

(previously identified as

rogB

, Gutekunst

et al.

, 2003), three genes encoding LPXTG-containing proteins (

gbs1478

,

gbs1477

and

gbs1474

) flanking the

srtC3

and

srtC4

genes. Note that in 2603V/R, a one-base-pair deletion inactivates

rogB

and that

gbs1477

is replaced by

sag1407

which encodes an homologous 75 kDa LPXTG protein (44% identity). In strain A909, the

srtC3-C4

locus as defined above is replaced by a segment containing two genes encoding LPXTG proteins (

sak1441

and

spb1

) and three genes encoding sortases (

sak1439 -1437

). However, only Sak1439 and Sak1437 possess the five critical amino acids (H, F, TC, R) required for sortase activity. In this figure, only the differences between the different loci are shown. Genes encoding sortase, regulatory and LPXTG proteins are drawn in yellow, red and blue respectively. The presence of the canonical pilin motif (Pm) and E-box (Eb) as defined by Ton-That

et al

. (2004) in the putative pilin proteins are depicted by a blue box. No pilin motif was found in Sak1441 and Spb1. The black lines below the genes in the srtC3-C4 locus of NEM316 delineate the in-frame deletions (∆) introduced to inactivate rogB, gbs1478, gbs1477, srtC3 and srtC4. The srtC3-srtC4 double mutant and the gbs1474 mutant were constructed by replacement of a chromosomal segment (deletion/insertion) with a kanamycin-resistance gene (kan). Gene nomenclature refers to strain NEM316 (gbs) (Glaser et al., 2002), strain 2603V/R (sag) (Tettelin et al., 2002), or strain A909 (sak) (Tettelin et al., 2005) sequence annotation.

4 S. Dramsi et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology

To determine if Gbs1477, Gbs1478 and Gbs1474 areconstituents of the same pilus structure, we carried out adouble-labelling experiment. WT bacteria were stainedwith mouse anti-Gbs1477 polyclonal antibodies (pAb) fol-lowed by 10 nm gold-labelled IgG and then with rabbitanti-Gbs1478 pAb followed by 20 nm gold-labelled IgG.Gbs1478 staining was detected at various locations of thepilus structure, including the base and the tip (Fig. 4A).A similar experiment was performed with mouse anti-Gbs1477 pAb and rabbit anti-Gbs1474 pAb. Gbs1474staining was mostly spotted at the base of the pilus(Fig. 4B) but was also seen at random along the pilusshaft (data not shown).

To understand better the molecular mechanisms ofpilus biogenesis, we conducted a systematic genetic anal-ysis by inactivating each structural gene of the srtC3-C4locus in strain NEM316. As shown in Figs 2 and 3, dele-tion of the gbs1477 gene encoding the putative pilinresulted in the loss of pilus-like structures at the bacterialsurface. In contrast, inactivation of the other LPXTG-encoding genes of the srtC3-C4 locus, i.e. gbs1478 andgbs1474, did not impede the synthesis of apparently nor-

Fig. 2. Immunolocalization of the pilin Gbs1477 and of the pilin-associated compo-nent Gbs1474 on GBS cell surface by SEM. S. agalactiae strain NEM316 and its derivative mutants were immobilized on carbon grids and stained with Gbs1477 (A) or Gbs1474 (B) spe-cific antisera and 10 nm colloidal gold-conju-gated anti-rabbit IgG antibody. Note that only about 10% of SrtA– cells expressed pili. Sam-ples were viewed by SEM. Scale bars, 0.1 µm.

Fig. 3. Immunogold TEM of pilus-like structures on the cell surface of S. agalactiae NEM316 and its derivative mutants. Immunogold labelling and TEM with Gbs1477-specific antiserum and 10 nm col-loidal gold-conjugated anti-rabbit IgG antibody.

Pili of Streptococcus agalactiae 5


mal pili (Figs 2 and 3). Deletion of both genes encodingsortases C (gbs1476 and gbs1475) resulted in completeloss of pilus-like structures at the bacterial surface, indi-cating that these enzymes are involved in pilus assembly(Figs 2 and 3). Surprisingly, deletion of the srtA gene

coding for the housekeeping sortase of S. agalactiae alsostrongly reduced pilus biosynthesis. We determined thatonly about 10% of SrtA– cells displayed pili whichappeared longer than those of the wild-type (WT) strain(Figs 2 and 3). In contrast, pili were never found at thesurface of the SrtC3C4– double mutant.

Role of SrtC in the polymerization of pilus components

Pilus biogenesis was studied by Western blotting of cellwall protein extracts using specific antibodies directedagainst the pilus-associated components. Antiseraraised against the major pilin Gbs1477 and the minorpilin-associated proteins Gbs1478 and Gbs1474 werefound to be highly specific as demonstrated by theabsence of reactive protein in the extracts from themutant strains Gbs1478–, Gbs1477– and Gbs1474–

(Fig. 5). The antiserum raised against the pilus compo-nent Gbs1478 recognizes a band of approximately100 kDa that corresponds to the predicted size ofGbs1478 monomer and high-molecular-weight speciesthat likely represent Gbs1478-containing polymers.These polymers were detected in the WT, RogB–, SrtC3–

and SrtC4– strains but not in the SrtC3C4–, Gbs1477–

and Gbs1474– mutants (Fig. 5A).

Fig. 5. Western blot analysis of cell wall-anchored proteins of S. agalactiae strains with anti-Gbs1478 (A), anti-Gbs1477 (B) and anti-Gbs1474 (C) antisera. Mutanolysin extracted surface proteins from S. agalactiae strain NEM316 and its isogenic derivatives mutants were separated on 3–8% gradient Tris-acetate Criterion XT SDS-PAGE gels and detected by immunoblotting with specific anti-Gbs1478, anti-Gbs1477 or anti-Gbs1474 antiserum. Monomers of each protein are indicated by a black arrow. The quantity loaded in each well is indicated below in µg.

Fig. 4. Localization of the pilus components of GBS. Double-labelling experiments were performed on the WT strain NEM316 with: (A) mouse anti-Gbs1477/IgG−10 nm gold-labelled cells subsequently stained with rabbit anti-Gbs1478/IgG−20 nm gold-labelled (the insert shows a high magnification view by SEM), and (B) mouse anti-Gbs1477/IgG−10 nm gold and rabbit anti-Gbs1474/IgG−20 nm gold-labelled IgG viewed by TEM.

6 S. Dramsi et al.


The antiserum specific for Gbs1477 recognized severalreactive species in the cell wall extracts of the differentstrains except in the mutant strain Gbs1477– (Fig. 5B).The band of about 75 kDa likely corresponds to Gbs1477monomer and the high-molecular-weight species toGbs1477-containing polymers. Whereas the hybridizationpatterns of cell wall extracts of WT, SrtC3– and SrtC4–

strains revealed the presence of high-molecular-weightpolymers containing Gbs1477, these products were notdetected in the extracts of the SrtC3C4– double mutant.Concomitantly, an increased quantity of Gbs1477 mono-mer was detected in the SrtC3C4– double mutant.Moreover, a significant increase of Gbs1477-containingpolymers was observed in the extracts of Gbs1478–

mutant strain (note the reduced quantity of cell wallextracts loaded for Gbs1478– mutant strain 0.2 µg versus5 µg). The hybridization pattern of cell wall extracts of theGbs1474– mutant was similar to that of the parental strain,although a reduction of Gbs1477 monomers in the cellwall fraction was noted compared with the parental strainNEM316.

A similar analysis was carried out with antibodiesdirected against Gbs1474. As shown in Fig. 5C, Gbs1474monomers (≈ 30 kDa) were readily detected in the cellwall fraction of the SrtC3C4–, whereas the parental strainand the other mutant strains exclusively contained thepolymer form of this protein. The results obtained in this analysis confirmed those obtained with the Gbs1478 and

Gbs1477 antisera showing the necessity of SrtC3 orSrtC4 in pilin polymerization and the absolute requi-rement of the major pilin component Gbs1477 for pilusbiogenesis.

Modulation of pili biosynthesis by SrtA

As previously mentioned, fewer pili were detected in theSrtA– mutant. Thus, production of the major pilin Gbs1477was studied by Western blotting of total protein extracts inthe various strains, including the SrtA– mutant. As shownin Fig. 6, expression of Gbs1477 was dramaticallyreduced in the SrtA– mutant. The effect of srtA inactivationwas even stronger on Gbs1477 expression compared withthat produced by the inactivation of the rogB activator.Similar results were obtained for Gbs1478 and Gbs1474(data not shown).

To investigate whether srtA inactivation affects gbs1477transcription, quantitative RT-PCR was performed on totalRNAs extracted from the WT NEM316, SrtA–, RogB– andGbs1478– mutants grown in Todd–Hewitt (TH) at 37°C(OD600 = 0.6). As shown in Fig. 7, a dramatic reduction ofgbs1477 transcripts were detected in the SrtA– mutantand similar results were obtained with gbs1478 andgbs1474 transcripts (data not shown). Interestingly, thesignificant reduction of gbs1477 transcript in the RogB–

mutant (but also of gbs1478 and gbs1474, not shown)

Fig. 6. Western blot analysis of total protein extracts of GBS strains. Total protein extracts of S. agalactiae strain NEM316 and its isogenic mutants were separated on 3–8% gradient Tris-acetate Criterion XT SDS-PAGE gels and detected by immunoblotting with specific anti-Gbs1477 antiserum. The last two lanes (noted IP) represent immu-noadsorptions performed on total protein extracts from the WT strain NEM316 using 2 µg of anti-Gbs1474 (α1474) or anti-Gbs1478 (α1478) pAb. Immunocomplexes were captured using protein G-Sepharose beads and analysed by immunoblotting with Gbs1477 antiserum (α1477). The composition of some polymers have been identified based on their apparent molecular weight and the immu-noreactive patterns of the proteins extracted from Gbs1474– and Gbs1478– mutants and of the complexes immunoabsorbed with α1474 and α1478 (m, monomer; d, dimer).

Fig. 7. Quantitative RT-PCR (qRT-PCR) analysis of gbs1477 and scpB gene expression in GBS strains. The isogenic SrtA– was previ-ously described (Lalioui et al., 2005). The qRT-PCR experiments were performed in quadruplets using two different RNA preparations. Sim-ilar results were obtained with the two preparations and the mean values of one experiment are shown here. The SD was calculated from the four experimental values of this analysis.



confirmed that RogB is a transcriptional activator of thesrtC3-C4 operon (Gutekunst et al., 2003). The geneencoding the classical LPXTG-containing protein ScpBknown to be a substrate for SrtA (Lalioui et al., 2005), wasused as a control. In these experiments, similar levels ofscpB transcripts were detected in the WT and SrtA–

strains (Fig. 7). DNA fragments were not amplified byPCR from GBS RNA in the absence of reverse tran-scriptase (data not shown), excluding the possibility ofDNA contamination in the RNA preparation. Decreasedexpression of the srtC3-C4 locus was by far more pro-nounced in the SrtA– mutant than in the RogB– mutant, afinding fully consistent with the Western blotting analysis.Lastly, this RT-PCR analysis showed that the increasedproduction of Gbs1477 in the Gbs1478– mutant, asrevealed by Western blotting, is due to an increasedamount of gbs1477 transcripts.

Gbs1477 interacts with Gbs1474 and Gbs1478

We investigated whether Gbs1477 could physically inter-act with Gbs1474 and Gbs1478 in total extracts of the WTstrain NEM316. Immunoadsorptions were performed ontotal protein extracts of exponentially growing cells using2 µg of either anti-Gbs1474 or anti-Gbs1478 pAb. Immu-nocomplexes were captured using protein G-Sepharosebeads and analysed by immunoblotting with anti-Gbs1477pAb. As shown in Fig. 6, Gbs1477 can interact with bothGbs1474 and Gbs1478, revealing the presence of thevarious combinations of dimers and trimers. Similar

immunoadsorptions performed on cell wall extracts of WT,SrtC3C4–, Gbs1478–, Gbs1477– and Gbs1474– strainsshowed that Gbs1474 and Gbs1478 interact withGbs1477 polymers (Fig. 8). No signal was detected in theGbs1477– mutant. In the Gbs1478– strain, the majorreactive band was of about 115 kDa corresponding to thesize of a Gbs1477 : Gbs1474 dimer. These resultsindicate that Gbs1477 polymers can specifically interactwith the two other pilus components, Gbs1474 andGbs1478.

Role of pili in adhesion to human epithelial cells

Many Gram-negative bacterial pathogens display long pilithat are necessary for adhesion and invasion of host tis-sues. We thus investigated whether GBS pili could beinvolved in adhesion to human pulmonary epithelial cellsA549. Cells were infected at a multiplicity of infection (moi)of 10 bacteria per cell and adherence frequencies werecalculated from the numbers of bacteria remainingattached to cells after the incubation period with respectto the total number of inoculated bacteria. The level ofadherence of the parental WT strain is arbitrarily reportedas 100 and the levels of adherence of the various isogenicmutants are relative values. As shown in Fig. 9, adher-ence of the Gbs1478– mutant was significantly reducedcompared with the parental strain, to a level similar to thatobtained with the SrtA– mutant (Lalioui et al., 2005). Incontrast, non-piliated mutants obtained either by deletingthe gene encoding the major pilin (Gbs1477– mutant) orby inhibiting the polymerization process (SrtC3C4–

mutant) were unaffected for adherence to A549 cells.

Fig. 8. Interaction of Gbs1474 and Gbs1478 with Gbs1477 polymers in cell wall extracts of GBS strains. Immunoadsorptions were per-formed on cell wall proteins extracted from exponentially grown bac-teria using 2 µg of either affinity-purified anti-Gbs1474 (α1474) or anti-Gbs1478 (α1478) pAb. Immunocomplexes were captured using protein G-Sepharose beads and analysed by immunoblotting with Gbs1477 antiserum (α1477). The composition of some polymers have been identified based on their apparent molecular weight and/or the immunoreactive patterns of the proteins extracted from WT and Gbs1478– strains (d, dimer; p, polymer).

Fig. 9. Adherence of GBS strains to human pulmonary epithelial cells A549. Cells were infected at a moi of 10 bacteria per cell for 1 h at 37°C, and adherence frequencies were calculated from the num-bers of bacteria remaining attached to cells after the incubation period with respect to the total number of inoculated bacteria. The level of adherence of the WT strain is arbitrarily reported as 100 and the levels of adherence of the various mutants are relative values. The results are presented as mean values (± SD) from at least three different experiments performed in duplicates.

8 S. Dramsi et al.


Intermediate levels of adherence were observed with theRogB– and Gbs1474– mutants (Fig. 9). Adherence of GBSisogenic mutants to A549 cells were also visualized byimmunofluorescence (Fig. S1). The binding capacity ofGbs1478– mutant to A549 cells was strongly reduced,much like the SrtA– mutant (A). This result was confirmedusing HeLa human epithelial cell line (B). Taken together,these results indicate that Gbs1478 is the pilin-associatedadhesin and demonstrate that the adhesin activity of thisprotein does not require the synthesis of the pilus.

Discussion

This manuscript reports new data on pilus biosynthesis inGBS, a major pathogen in neonates. Pili or fimbriae arelong, filamentous, multimeric macromolecules found onthe bacterial surface. Pili in Gram-negative bacteria areinvolved in various attachment processes (Gilsdorf et al.,1997; Nassif et al., 1997; Sauer et al., 2000; Chiavelliet al., 2001). In contrast, only few Gram-positive bacteriahave been reported to elaborate pili, mainly in bacteria ofthe oral flora (Actinomyces spp., Streptococcus parasan-guinis or Streptococcus salivarius). Pili in Gram-positivebacteria are usually shorter and thinner than those ofGram-negative bacteria (for a review, see Wu and Fives-Taylor, 2001). Actinomyces naeslundii, present in thehuman dental plaque, forms two types of antigenic andfunctionally distinct fimbriae, type 1 and type 2. Type 1fimbriae (FimP) mediate adhesion to immobilized proline-rich proteins of saliva (Gibbons et al., 1998). Type 2 fim-briae (FimA) are responsible for the lactose-sensitive co-aggregation of A. naeslundii with oral streptococci andinteractions with various mammalian cells (Brennan et al.,1986; McIntire et al., 1988). Actinomyces species elabo-rate fimbriae that are composed of LPXTG-containingsubunit proteins (Yeung and Cisar, 1990). Knockout muta-tions of A. naeslundii sortase homologues abolish fimbrialassembly (Yeung and Ragsdale, 1997). Theses sortasesbelong to the class C according to our recent nomencla-ture (Dramsi et al., 2005).

In the present work, we report that S. agalactiae strainNEM316 possesses two gene clusters, srtC1-C2 andsrtC3-C4 coding for pilus biosynthesis, that are almostidentical to those found in strain 2603V/R (Fig. 1). Usingelectron microscopy and immunogold labelling, weshowed that strain NEM316 assembles pili only from thesrtC3-C4 locus. In contrast, it was recently shown thatGBS strain JM9130013 expresses pili from the srtC1-C2locus (sag0644-0649; Lauer et al., 2005). This variabilityin srtC locus expression is likely due to the presence offrameshift mutations in their specific regulatory genes,sag0644 or rogB (Fig. 1). These regulatory genes codefor transcriptional activators belonging to different families,which suggests that their expression might be triggered

by different environmental signals. Sag0644 contains twoAraC-like DNA binding domains in the C-terminus and anN-terminal dimerization and/or ligand binding domain.Signalling molecules of AraC family are normally smallcompounds such as sugars (Ara for arabinose), but pro-teins also have been shown to modulate AraC function(Martin and Rosner, 2001; Plano, 2004). RogB is a mem-ber of the RofA-like regulators (also known as RALP)described in both Streptococcus pyogenes and Strepto-coccus pneumoniae (Kreikemeyer et al., 2003). RogB hasbeen previously identified in GBS strain 6313, where itpositively regulates the transcription of gbs1478 andgbs1477 and of other virulence genes (Gutekunst et al.,2003). The RogB– mutant in strain 6313 exhibited areduced adherence to human epithelial cells (Gutekunstet al., 2003) and, consistently, we observed that inNEM316 RogB activates the expression of the srtC3-C4operon which includes the gene for the adhesin Gbs1478(Fig. 7). Possible co-ordinately regulated expression ofeach srtC locus with a specific set of virulence genes isan attractive hypothesis that will be tested in future exper-iments. Of interest, the 12 kb pathogenicity island presentin the virulent S. pneumoniae strain TIGR4 includes sixgenes encoding three LPXTG proteins and three sortasesof class C respectively, which are regulated by RlrA, atranscriptional regulator of the RALP family (Hava et al.,2003). Moreover, one of the LPXTG proteins (SP0463)possesses the canonical pilin motif, suggesting that thisS. pneumoniae pathogenicity island is also involved inpilus biosynthesis. This hypothesis has been recently con-firmed (Barocchi et al., 2006; Lemieux et al., 2006).

A detailed structural and functional analysis of thesrtC3-C4 locus in GBS strain NEM316 enabled us tocharacterize the role of three precursor proteins,Gbs1478, Gbs1477 and Gbs1474, in pilus biogenesis.Immunogold staining revealed that Gbs1477 is distributeduniformly along pili and constitutes the core of the pilus.In contrast, Gbs1478 and Gbs1474 are minor pilus-associated components randomly distributed in thepilus including the base (Gbs1474 and Gbs1478) and thetip (Gbs1478) (Figs 2 and 4). Adherence assays tohuman epithelial cells unambiguously demonstrated thatGbs1478 is an adhesin. Taken together, these resultsstrongly suggest that GBS pili are composed of threeprecursor proteins: Gbs1477, the bona fide pilin is themajor pilus component; Gbs1474, a minor pilus-associated component that may anchor the pilus to thecell wall, and Gbs1478, the pilus-associated adhesin.Hence, the pilus of GBS strikingly resembles that previ-ously described in C. diphteriae (Ton-That and Schnee-wind, 2004). Assembly of this type of pilus is a complexprocess involving at least two main steps: (i) polymeriza-tion, i.e. the covalent anchoring of Gbs1477 pilin subunits,which we have shown to be dependent on SrtC and (ii)



anchoring of the pilus to the cell wall which requires themajor SrtA.

Production of the LPXTG-containing proteins encodedby the srtC3-C4 locus is dependent on SrtA, as the pilicomponents were not produced in the SrtA– mutant. Thisregulation likely occurs at the transcriptional level, as aqRT-PCR analysis showed that inactivation of srtAstrongly diminishes the levels of gbs1477 , gbs1478 andgbs1474 transcripts (Fig. 7 and data not shown). It isapparently specific to srtC3-C4 mRNAs, as we demon-strated that transcription of the scpB gene encoding theC5a peptidase, a canonical SrtA substrate (Lalioui et al.,2005), was not altered in the SrtA– mutant (Fig. 7). Basedon these results, we suggest that pilus synthesis requiresa ‘sorteome’ containing SrtA and SrtC sortases and thatabsence of SrtA leads to a specific degradation of themRNAs encoding the SrtC substrates by an as yetunknown mechanism. We also demonstrated that the syn-thesis of a structurally normal pilus requires only the majorpilin component. However, closer examination of the EMmicrographs revealed that slightly longer pili are presentat the surface of the adhesin mutant Gbs1478–. Of note,in this mutant, the Gbs1477 pilin is overproduced, whichcould account for the presence of longer pili. Consistently,overexpression of the major pilin gene sag0649 in GBSstrain COH1 resulted in the production of longer pili (Laueret al., 2005). These results suggest that the stoichiometryof the different pilus components could modulate the piluslength, a model that will be tested in future experiments.The minor component Gbs1478 in the pilus confers itsadhesive property, i.e. adherence to human epithelialcells. However, as this adhesin is not required for pilussynthesis, we could not discriminate between a modelinvolving an early incorporation of this protein at the initi-ation step or a late incorporation in a formed pili. Theapparently paradoxical situation of a pilus that carries theadhesive property and yet is dispensable for binding wasreported previously in the Escherichia coli Pap pilus modelsystem (Lindberg et al., 1986). We therefore postulate thatin the absence of pilus, Gbs1478 behaves as a canonicalLPXTG-containing protein that is anchored to the cell wallby SrtA, where it can also mediate adherence to culturedepithelial cells. Indeed, in the Gbs1477– mutant, Gbs1478is found essentially as a monomer or a dimer withGbs1474 in the cell wall extracts (Fig. 5). We also con-firmed by IEM that Gbs1478 could be detected on thebacterial surface in the absence of pilus structures (datanot shown). These results demonstrate that pili in impor-tant Gram-positive pathogens, play a central role in host–pathogen interactions.

The NEM316 srtC1-C2 locus (gbs0626 to gbs0634) ispart of genomic island VI located in the close vicinity ofthe cyl operon (gbs0644 to gbs0655) involved in virulence.In contrast, the srtC3-C4 locus is located in the genome

backbone. DNA-array hybridizations of a collection ofGBS strains of human and animal origins showed thatboth SrtC encoding loci exhibit considerable variation(Brochet et al., 2006). Of note, three out of the four pro-tective antigens present in the universal GBS vaccinerecently proposed (Maione et al., 2005) were LPXTG-containing proteins encoded by the srtC1-C2 (Sag0645and Sag0649) and the srtC3-C4 loci (Sag1408/gbs1478).One might speculate that this vaccine will likely selectGBS variants expressing new pili antigens.

Experimental procedures

Bacterial strains, media and growth conditions

Streptococcus agalactiae NEM316 was responsible for a fatalsepticaemia and belongs to the capsular serotype III. Thecomplete genome sequence of this strain has been deter-mined (Glaser et al., 2002). E. coli DH5α (Gibco-BRL) wasused for cloning experiments. E. coli BL21/pDIA17 (Munieret al., 1991) was used for expression of recombinantproteins. S. agalactiae was cultured in TH broth or agar(Difco Laboratories, Detroit, MI) and E. coli in Luria–Bertanimedium. Unless otherwise specified, antibiotics were usedat the following concentrations: for E. coli – ampicillin,100 µg ml−1; erythromycin, 150 µg ml−1; kanamycin, 50 µgml−1; for S. agalactiae – erythromycin, 10 µg ml−1; kanamycin,1000 µg ml−1. S. agalactiae liquid cultures were grown at37°C in standing filled flasks. The various strains andplasmids are shown in Table 1.

General DNA techniques

Standard recombinant techniques were used for nucleic acidcloning and restriction analysis (Sambrook et al., 1989).Plasmid DNA from E. coli was prepared by rapid alkalinelysis using the Nucleospin Plasmid kit (Macherey-Nagel).Genomic DNA from S. agalactiae was prepared using theBoehringer-Roche High Purification kit. PCR was carried outwith Ampli Taq Gold polymerase as described by the manu-facturer (Applied Biosystems). Amplification products werepurified on Sephadex S-400 columns (Pharmacia) andsequenced with an ABI 310 automated DNA sequencer,using the ABI PRISM dye terminator cycle sequencing kit(Applied Biosystems).

Construction of deletion mutants

The primers used for the construction of deletion alleles arelisted in Table S1. In-frame deletions in gbs1479 (O1-O2;O3-O4), gbs1478 (O5-O6; O7-O8), gbs1477 (O9-O10;O11-O12), gbs1476 (srtC3) (O17-O18; O19-O20), gbs1475(srtC4) (O21-O22; O23-O24) were constructed by usingsplicing-by-overlap-extension PCR. The srtC3-C4 doublemutant (O25-O26; O27-O28) and the gbs1474 (O13-O14;O15-O16) mutant were constructed as previously described(Lalioui et al., 2005) by inserting a kanamycin-resistancegene. To carry out chromosomal gene inactivation, appropri-ate PCR fragments were cloned into the thermosensitive

10 S. Dramsi et al.


shuttle plasmid pG+host5. Electroporation of GBS strainsand allelic exchange were performed as previously described(Biswas et al., 1993). In-frame deletions or insertional/inacti-vation of all genes were confirmed by PCR and sequenceanalysis.

Real-time quantitative PCR

Primers were designed with BEACON Designer software andare listed in Table S1. RNA (15 µg) was treated with TurboDNase for 30 min at 37°C according to the recommendationsof the manufacturer (Turbo-DNA-free kit from Ambion).DNAse was removed with DNase inactivation reagent pro-vided in the kit and by applying the sample on AmiconUltrafree-DA filter. cDNA synthesis was performed on DNase-treated RNA (5 µg) with random hexamers (Roche) usingSuperscript II RT (Invitrogen) according to the protocol rec-ommended by the manufacturer. Real-time quantitative PCRwas performed in a 25 µl reaction volume containing cDNA,12.5 µl iQ™ SYBR Supermix (Bio-Rad) and 0.5 µl gene-specific primer (10 µM). Amplification and detection of spe-cific products were performed with the MyiQ Single-colourreal-time PCR detection system (Bio-Rad) with the followingcycle profile: one cycle at 95°C for 3 min followed by 40cycles at 95°C for 15 s, 55°C for 15 s and 72°C for 10 s. Thespecificity of the amplified product was verified by generatinga melting curve with a final step of 80 cycles of 10 s at aninitial temperature of 55°C, increasing 0.5°C each cycle upto 95°C. Loss of fluorescence was observed at the denatur-ing/melting temperature of the product (Ririe et al., 1997). Tocheck whether contaminating chromosomal DNA waspresent, each sample was tested in control reactions that didnot contain reverse transcriptase. For each condition, qua-druple assays were done. The analysis gave a threshold (CT)value for each sample, which is defined as the cycle at whicha significant increase in amplification product occurs. Themean CT value was calculated for each quadruple reaction.

A ∆CT value was then calculated for each sample by sub-stracting the mean CT value of the target gene from the meanCT value of the rpoB reference gene (rpoB encodes theessential and constitutively expressed β-subunit of RNA poly-merase). The data were transformed from an exponential toa linear scale by using the formula × = 2–∆CT (Livak andSchmittgen, 2001).

Total and cell wall protein extracts

Bacteria were grown in TH medium at 37°C and harvestedfor protein analysis during late exponential phase of culture.Bacteria were washed twice in Tris-HCl buffer (50 mM,pH 7.3) and once in the mutanolysin digestion buffer(Tris-HCl 50 mM Tris-HCl, pH 7.3 supplemented with 20%sucrose and 2 mM AEBSF). Mutanolysin (Sigma), dissolvedto 5000 U ml−1 in potassium phosphate buffer (10 mM,pH 6.2), was then added to the bacterial suspension to givea final concentration of 100 U ml−1. The digestion wasperformed for 5 h at 37°C under gentle agitation. Aftercentrifuging at 13 000 g for 15 min at 4°C, supernatants cor-responding to the cell wall fractions were analysed on SDS-PAGE or kept frozen at −20°C.

Total proteins extracts from bacteria were prepared usingFastProtein Blue (Bio 101 Systems) according to the manu-facturer’s recommendations. S. agalactiae were disruptedusing the FastPrep Instrument at a speed of 6.5 for 40 s.

Immunoblots and immunofluorescence staining

Generation of pAb against recombinant 6xHis-Gbs1478,6xHis-Gbs1477 and 6xHis-Gbs1474 were performed aspreviously described (Lalioui et al., 2005). For analysis ofGbs1478, Gbs1477 and Gbs1474 expression, bacterial pro-teins were separated into cell wall and total protein extracts(see above) Proteins were boiled in Laemmli sample buffer,

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid Relevant propertiesa Source or reference

StrainsEscherichia coli

DH5α recA1 gyrA (Nal), ∆(lacIZYA-argF)[Φ80∆lac∆(lacZ)M15] Gibco-BRLBL21λDE3 F-ompT gal (dcm) (lon) hsdSB(rB

– mB–) endA1 hsdR17(rK

–mK+) Studier and Moffatt (1986)

Streptococcus agalactiaeNEM316 Serotype III isolated from neonate blood culture (early onset disease) Gaillot et al. (1997)NEM2135 NEM316∆srtA (Km; SrtA–) Lalioui et al. (2005)NEM2396 NEM316∆gbs1479 (in frame deletion) This workNEM2397 NEM316∆gbs1478 (in frame deletion) This workNEM2398 NEM316∆gbs1477 (in frame deletion) This workNEM2399 NEM316∆srtC3 (in frame deletion) This workNEM2400 NEM316∆srtC4 (in frame deletion) This workNEM2401 NEM316∆srtC3-C4 (Km) This workNEM2402 NEM316∆gbs1474 (Km) This work

PlasmidspG+host5 Em; ColE1 replicon, thermosensitive derivative of pGK12; MCS pBluescript Biswas et al. (1993)pDIA17 Cm, oriR pACYC184, Tet promoter ∆lacI Munier et al. (1991)pIVEX2.4b-NdeI Ap, oriR pUC, T7 promoter, His-Tag coding sequence Roche Applied Science

a. Ap, Cm, Em, Km, Nal, Sp, Str (or Sm) and Tc are resistance to ampicillin, chloramphenicol, erythromycin, kanamycin, nalidixic acid,spectinomycin, streptomycin and tetracycline respectively. MCS, multiple cloning site.



resolved on Tris-Acetate Criterion XT gradient gels 3–8%SDS-PAGE gels and transferred to nitrocellulose membrane(Hybond-C, Amersham). Gbs1478, Gbs1477 or Gbs1474was detected using specific pAb and horseradish peroxidase(HRP)-coupled anti-rabbit secondary antibodies (Zymed) andthe Western pico chemiluminescence kit (Pierce).

Expression and purification of recombinant 6xHis-Gbs1478 , 6xHis-Gbs1477 and 6xHis-Gbs1474

DNA fragments intragenic to gbs1478, gbs1477 and gbs1474were produced by PCR using genomic DNA of NEM316 astemplate and the primers O29-O30, O31-O32 and O33-O34respectively (Table S1). These DNA fragments were digestedwith the appropriate enzymes (NcoI or NdeI and BamHI) andcloned into pIVEX 2.4b-NdeI (Novagen). The resulting plas-mids were introduced into E. coli DH5α for sequence analysisand in E. coli strain BL21/pDIA17 for protein expression.Recombinant 6xHis-Gbs1478, 6xHis-Gbs1477 and 6xHis-Gbs1474 were purified under denaturing conditions by affin-ity chromatography on Ni-NTA columns according to themanufacturers’ recommendations (Novagen). Protein puritywas checked on SDS-PAGE and accurate protein concentra-tions were determined with the BCA system (Pierce).

Generation of rabbit and mice pAb

To generate antibodies, each rabbit was first tested for theabsence of GBS cross-reactivity (pre-immune serum) andthen immunized with 500 µg of each protein mixed in Freud’scomplete adjuvant, followed by two booster injections after3 weeks, each with 500 µg of protein mixed with Freund’sincomplete adjuvant. Three weeks after the third injection, theanimals were euthanized and exsanguinated by cardiacpuncture. The specificity of each antibody was determined byimmunoblotting against purified antigen as well as crudestreptococcal cell extracts prepared from WT and mutantstrains.

For double-labelling experiments, pAb against Gbs1477were generated in mice using a very similar procedure exceptfor the quantity of the antigen injected: 50 µg per boost/mouse.

Cell culture techniques and adherence assays

The human cell lines A549 (ATCC CCL-185) from an alveolarepithelial carcinoma and HeLa (ATCC CCL-2) from a cervixcarcinoma, were cultured in Dulbecco’s modified Eaglemedium (DMEM) containing Glutamax (Invitrogen) supple-mented with 10% fetal calf serum (FCS). Cells were incu-bated in 10% CO2 at 37°C and were seeded at a density of2–5 × 105 cells per well in 24 well tissue culture plates. Mono-layers were used after 24–48 h of incubation.

Bacteria were grown to mid-log phase in TH broth to anOD600 of 0.4 (approximately 108 cfu ml−1), washed once inPBS and resuspended in DMEM. Cells were infected at a moiof 10 bacteria per cell for 2 h at 37°C in 10% CO2. Themonolayers were then washed four to five times with PBS,and the cells were disrupted by the addition of 1 ml of sterile

deionized ice-cold water and repeated pipetting. Serial dilu-tions of the lysate were plated onto TH agar for count of viablebacteria. The per cent of adherence was calculated as fol-lows: (cfu on plate count/cfu in original inoculum) × 100.Assays were performed in duplicate and were repeated atleast three times.

Immunogold electron microscopy

For scanning electron microscopy (SEM) analysis, bacteriawere applied to glass coverslips, and fixed with 0.1% glut-araldehyde/4% paraformaldehyde in 0.1 M Sorensen buffer(pH 7.2) for 30 min. Fixed bacteria were incubated in PBSsupplemented with 0.25% NH4Cl for 20 min then washedextensively with PBS. Samples were incubated in ProteinBlock (Aurion) for 30 min. Following incubation for 1 h withthe primary antibody, samples were washed and incubatedfor an additional hour with the secondary antibody conju-gated to 10 nm colloidal gold. Preparations were washedwith PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacody-late buffer (pH 7.2) overnight at 4°C, then washed threetimes for 5 min (each time) in 0.2 M cacodylate buffer, post-fixed for 1 h in 1% osmium in 0.2 M cacodylate buffer andrinsed with distilled water. Bacteria were dehydrated througha graded series of ethanol (25%, 50%, 75%, 95% and 100%)followed by critical point drying with CO2. Dried specimenswere sputter coated twice with carbon, with a BALTEC MED010 evaporator and were examined and photographed witha JEOL JSM 6700F field emission scanning electron micro-scope operating at 5 or 7 kV. Images were acquired simulta-neously from the upper SE detector and the YAG BSEdetector.

For transmission electron microscopy (TEM) analysis,bacteria were washed once in PBS, fixed with 0.1%glutaraldehyde/4% paraformaldehyde in 0.1 M Sorensenbuffer (pH 7.2) and placed on an N Formvar grids for 20 min.Fixed bacteria were incubated in PBS supplemented with0.25% NH4Cl for 20 min then washed extensively with PBS.Samples were incubated in Protein Block (Aurion) for 30 min.Following incubation with the primary antibody (1/100) for1 h, samples were washed and incubated for an additionalhour with the secondary antibody conjugated to 10 nm col-loidal gold. Grids were washed twice with PBS-BSA andbacteria coupled with antisera were fixed with 1% glutaralde-hyde for 20 min. Following three washes in sterile water, gridswere stained with 2% uranyl acetate for 30 s, and observa-tions were performed by TEM using a JEOL 1200 EX at 80 kVand a CCD camera (Megaview, Eloise).

For double-labelling experiments, the same procedure wasapplied using the mouse polyclonal α-Gbs1477 (1/100)revealed with 10 nm gold-goat anti-mouse IgG followed bythe rabbit polyclonal α-Gbs1478 (1/10) or α-Gbs1474 (1/100)revealed with 20 nm gold-goat anti-rabbit IgG.

Acknowledgements

We acknowledge the PFID for imaging of the adherenceassays (Fig. S1). We are extremely grateful to Farida Natofor immunization of mice with recombinant 6xHis Gbs1477and recovery of pAb directed against the major pilin, Olivier

12 S. Dramsi et al.


Poupel for invaluable help in RNA preparation and RT-PCRanalysis, Adeline Mallet for double-labelling experiments inSEM, Mathieu Brochet and Philippe Glaser for helpful discus-sions and sharing of unpublished data. We thank OlivierGaillot and Tarek Msadek for critical reading of the manu-script. This work was supported by research funds from theInstitut Pasteur [GPH N°9 and the Centre National de laRecherche Scientifique (CNRS)].

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Supplementary material

The following supplementary material is available for thisarticle online:Fig. S1. Immunofluorescence analysis of GBS adherence toA549 cells (A) and HeLa cells (B). Bacteria were revealedwith specific anti-NEM316 pAb and revealed with anti-rabbitIgG coupled to Alexa 546 or 488 respectively. Cellular F-actinwas visualized with phalloidin coupled to FITC or TRITC andnuclei were stained with DAPI (shown in blue).Table S1. Oligonucleotides used in this study.

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

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