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The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates

The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in... B r i e f D e f i n i t ive R e p o r t The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates 1,2,3 4,5 1,2 Asmaa Tazi, Olivier Disson, Samuel Bellais, 1,2 3 6 Abdelouhab Bouaboud, Nicolas Dmytruk, Shaynoor Dramsi, 6 7 8 Michel-Yves Mistou, Huot Khun, Charlotte Mechler, 1,2 6 4,5,9 Isabelle Tardieux, Patrick Trieu-Cuot, Marc Lecuit, 1,2,3,6 and Claire Poyart Institut Cochin, Université Paris Descartes Faculté de Médecine, Centre National de la Recherche Scientifique (UMR 8104), 75014 Paris, France Institut National de la Santé et de la Recherche Médicale, U1016, 75014 Paris, France Assistance Publique Hôpitaux de Paris, Service de Bactériologie, Centre National de Référence des Streptocoques, Hôpital Cochin, 75014 Paris, France Institut Pasteur, Groupe Microorganismes et Barrières de l’Hôte, 75015 Paris, France Institut National de la Santé et de la Recherche Médicale Avenir U604, 75015 Paris, France Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram Positif, URA Centre National de la Recherche Scientifique 2172, 75015 Paris, France Institut Pasteur, Unité d’Histotechnologie et Pathologie, 75015 Paris, France Assistance Publique Hôpitaux de Paris, Service d’Anatomie Pathologique, Hôpital Louis Mourier, 92700 Colombes, France Université Paris Descartes Faculté de Médecine, Assistance Publique-Hôpitaux de Paris, Service des Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants Malades, 75015 Paris, France Streptococcus agalactiae (group B streptococcus; GBS) is a normal constituent of the intestinal microflora and the major cause of human neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-onset disease (LOD), which is characterized by meningitis in infants after the first week of life. The pathophysiology of LOD remains poorly understood, but our epidemiological and histo- pathological results point to an oral route of infection. Here, we identify a novel ST-17– specific surface-anchored protein that we call hypervirulent GBS adhesin (HvgA), and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered more efficiently to intestinal epithelial cells, choroid plexus epithelial cells, and microvascular endothelial cells that constitute the blood–brain barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in nonadhesive bacteria conferred the ability to adhere to intestinal barrier and BBB-constituting cells. In orally inoculated mice, HvgA was required for intestinal colonization and translocation across the intestinal barrier and the BBB, leading to meningitis. In conclusion, HvgA is a critical virulence trait of GBS in the neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies. CORRESPONDENCE Claire Poyart: [email protected] Group B streptococcus (GBS; Streptococcus agalac- improvement in neonatal intensive care, up to OR tiae) is a Gram-positive encapsulated commensal 10% of neonatal GBS infections are lethal, and Marc Lecuit: bacterium of the human intestine that is also pres- 25–35% of surviving infants with meningitis [email protected] ent in the vagina of 15–30% of healthy women. experience permanent neurological sequelae Abbreviations used: BBB, blood– In neonates, it may turn into a deadly pathogen, (Edwards and Baker, 2005). GBS is also a brain barrier; CNS, central and it is the leading cause of neonatal pneumonia, nervous system; EOD, early-onset septicaemia, and meningitis (Edwards and Baker, disease; GBS, group B strepto- © 2010 Tazi et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after coccus; HvgA, hypervirulent GBS 2005). Despite early antimicrobial treatment and the publication date (see http://www.rupress.org/terms). After six months it is adhesin; LOD, late-onset disease; available under a Creative Commons License (Attribution–Noncommercial–Share qRT-PCR, quantitative RT-PCR; Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ TH, Todd Hewitt. M. Lecuit and C. Poyart contributed equally to this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 207 No. 11 2313-2322 www.jem.org/cgi/doi/10.1084/jem.20092594 The Journal of Experimental Medicine significant cause of morbidity and mortality in nonpregnant We show that this protein which we have called hypervirulent adults, particularly those with underlying diseases and the GBS adhesin (HvgA) mediates GBS neonatal intestinal coloniz- elderly (Phares et al., 2008). ation and crossing of the intestinal and blood–brain barriers, Two distinct GBS-associated clinical syndromes, referred leading to meningitis, which are key features of LOD. to as early-onset disease (EOD) and late-onset disease (LOD) have been recognized in neonates in their r fi st week of life (age RESULTS AND DISCUSSION 0–6 d) and after (age 7–89 d), respectively (Edwards and Baker, Epidemiological evidence that the ST-17 hypervirulent GBS 2005). Although intrapartum antibioprophylaxis for parturient clone is associated with LOD and neonatal meningitis women at risk for GBS infection has markedly decreased the We first analyzed 651 GBS isolates referred to the French na - incidence of EOD, it did not change that of LOD (Poyart et al., tional reference center for streptococci between 2006 and 2008; CDC, 2009). Epidemiological data collected worldwide 2009 from consecutive cases of invasive infection in neonates have shown that a substantial proportion of EOD and the ma- (meningitis, n = 138; bacteremia, n = 166) and in adults (men- jority of LOD are associated with capsular serotype III (Lin et al., ingitis, n = 16; bacteremia, n = 331). Serotype III accounts for 2006; Gherardi et al., 2007; Phares et al., 2008; Poyart et al., 86.2% of strains isolated from cases of neonatal meningitis 2008; CDC, 2009). Strains of serotype III contain a limited and 60.8% of neonatal bacteremia, but only 25.7% of bacte- number of clonal complexes, den fi ed by multilocus sequence remia in adults (Table I). Serotype III is significantly associ - typing. Among them, the ST-17 sequence type is strongly associ- ated with meningitis during EOD (79.3%; P < 0.0001) and ated with neonatal meningitis and was therefore designated as LOD (88%; P < 0.0001; Table I). Moreover, the serotype III “the hypervirulent clone,” despite the absence of experimental ST-17 clone is significantly associated with meningitis during data to support this assertion (Musser et al., 1989; Jones et al., EOD (79.3%; P < 0.0001) and LOD (82.6%; P < 0.0001), 2003, 2006; Brochet et al., 2006; Lamy et al., 2006; Bohnsack and with bacteremia during LOD (78.1%; P < 0.0001; Table I). et al., 2008; Poyart et al., 2008; Manning et al., 2009). In contrast, the ST-17 clone represents <12% of isolates from For EOD, the mode of transmission in newborns is thought adult patients with bacteremia (Table I). Together, these results to be vertical, by inhalation of GBS-contaminated amniotic or obtained from a total of 651 clinical strains demonstrate that vaginal fluid during parturition, followed by bacterial trans - ST-17 GBS strains account for >80% of neonatal meningitis, location across the respiratory epithelium and subsequent sys- strongly suggesting an enhanced virulence of the ST-17 clonal temic infection (Edwards and Baker, 2005). In contrast, for complex in the neonatal context. These epidemiological ob- LOD, the mode of transmission and the infection route remain servations thus prompted us to search for specific virulence elusive, although mother-to-child transmission might also be factors of the ST-17 clone that may account for its apparent involved. A plausible scenario would involve early intestinal higher pathogenicity in neonates, its close association with colonization by GBS that would lead in the r fi st days of life to LOD, and its meningeal tropism. its intraluminal intestinal multiplication, translocation across the intestinal epithelium, and access to the bloodstream. Histopathological study of a fatal case Indeed, an intestinal portal of entry for LOD is supported by of ST-17–associated LOD several lines of evidence: (a) 60 and 40% of the neonates asymp- A term female infant (gestational age, 39 wk; birth weight, tomatically colonized with GBS at birth remain positive for 3,140 g) was born by spontaneous vaginal delivery without bacteria at the rectal level at 4 and 12 wk of life, respectively complication. Maternal vaginal swab at 37 wk of gestation (Weindling et al., 1981); and (b) a longitudinal study of GBS was negative for GBS. There was no premature membrane vaginal and rectal colonization in women during and after rupture and neither skin nor rectal swab of the neonate was pregnancy has revealed that carriers are usually colonized for made at delivery. The mother and her breastfed baby were up to 2 yr by a single clone, which is also frequently found in discharged on day 4. On day 14 of life, the neonate developed newborn feces for up to 1 yr (Hansen et al., 2004). muscular hypotonia, poor suckling, hyperexcitability, and Once translocated in the bloodstream, GBS has the ability fever. Cerebrospinal fluid and blood cultures were positive for to cross the blood–brain barrier (BBB) and cause meningitis. GBS, which was later shown to belong to serotype III and Several virulence factors contribute to the pathogenesis of GBS clonal complex ST-17. Breast milk was not cultivated. Despite meningitis in animal models, but nearly all of them are in- adequate antimicrobial treatment associating amoxicillin, cef- volved in the septicemia phase of the infection, but not in GBS triaxone, and gentamicin, she died 8 h later and an autopsy adhesion to and crossing of the BBB (Maisey et al., 2008). One was performed. Cultures of stool, blood, and cerebrospinal fluid, exception is Srr-1, a recently characterized surface glycoprotein as well as colonic and brain autopsic tissue samples, were all that promotes adhesion to and invasion of human brain micro- positive for GBS. Immunohistochemistry of paran ffi -embedded vascular endothelial cells and contributes to BBB crossing in gut tissue samples led to the detection of GBS associated with mice (van Sorge et al., 2009). This illustrates that more studies the intestinal tissue and inside the lamina propria (Fig. 1, a and b). are needed to identify virulence factors of GBS, especially in GBS also heavily infected meningeal tissues, with intense in- regard to its meningeal tropism and its ability to trigger LOD. flammation indicated by the massive recruitment of poly - Here, we have identie fi d a novel ST-17–specic fi surface- morphonuclear cells (Fig. 1, c and d). GBS was also observed anchored protein, which is highly prevalent in cases of LOD. to be tightly associated with brain microvessel endothelial 2314 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Table I. Serotype and ST-17 distribution of 651 GBS strains isolated from neonatal and non-pregnant adult invasive infections in France between 2006 and 2009 Type of infection Serotype no. ST-17 no. Ia Ib II III IV V Total % % Neonatal meningitis 12 (8.7) 4 (2.9) 0 119 (86.2) 0 3 (2.1) 138 (100) 113 (81.9) EOD (≤6 d) 6 (20.7) 0 0 23 (79.3) 0 0 29 (100) 23 (79.3) LOD (≥7–89 d) 6 (5.5) 4 (3.7) 0 96 (88.1) 0 3 (2.7) 109 (100) 90 (82.6) Neonatal bacteremia 34 (20.5) 5 (3) 6 (3.6) 101 (60.8) 3 (1.8) 17 (10.2) 166 (100) 89 (53.6) EOD (≤6 d) 27 (29) 3 (3.2) 5 (5.4) 41 (44.1) 2 (2.2) 15 (16.1) 93 (100) 32 (34.4) LOD (≥7–89 d) 7 (9.6) 2 (2.7) 1 (1.4) 60 (82.2) 1 (1.4) 2 (2.7) 73 (100) 57 (78.1) Adult bacteremia 71 (21.4) 36 (10.3) 37 (11.2) 85 (25.7) 17 (5.1) 85 (25.7) 331 (100) 37 (11.2) Adult meningitis 4 (25) 2 (12.5) 1 (6.25) 8 (50) 0 1 (6.25) 16 (100) 5 (31.3) cells and choroid plexus epithelial cells, which constitute the HvgA is an ST-17–specific surface-anchored protein blood–brain parenchyma and blood–cerebrospinal fluid bar - that is overexpressed in vivo riers, respectively (Fig. 1, e–l). These bacteriological and histo- We first analyzed whether the ST-17 clone expresses specific pathological analyses of this fatal case of LOD are consistent surface-exposed molecules that could account for enhanced with the hypothesis that LOD results from the ability of GBS adhesive properties. The comparative analysis of GBS whole- ST-17 to efficiently colonize the intestine, cross the intestinal genome sequences has pinpointed several genes encoding surface components specic fi to the ST-17 clone (Tettelin et al., barrier, and cross the BBB. 2005; Brochet et al., 2006). In partic- ular, we have identie fi d mosaic variants at a single genomic locus (Lamy et al., 2006) encoding a cell wall–anchored protein, with two main variants dis- playing 38% overall amino acid iden- tity, namely Gbs2018A, which is also referred to as BibA (Santi et al., 2007), and Gbs2018C, which we have shown to be strictly specific to the “hyper- virulent” ST-17 clone (Lamy et al., 2006). These genes have conserved reg- ulatory regions and encode proteins with conserved N- and C-terminal parts, but a distinct central core. Indeed, comparison of the nucleotide sequences of the two loci has revealed that only the 5 and 3 ends of the two genes are highly conserved, displaying >90% sequence identity, whereas their in- ternal parts display low level (50–60%) Figure 1. ST-17 GBS crossing of the intestinal and BBBs in a fatal case of human neonatal LOD with meningitis. Immunohistological study of the intestine and the CNS of a fatal case of ST-17 LOD. Bacteria were labeled with a specific polyclonal anti - body to GBS and appear in reddish brown. Sections were counterstained with hematoxy- lin. GBS is present in the intestine (a and b), in meninges (c and d), in brain microvessels (e–j), as well as in choroid plexuses (k and l). JEM VOL. 207, October 25, 2010 2315 Figure 2. HvgA is a cell surface protein of GBS ST-17. (a) Structure of the bibA/hvgA locus in GBS strains NEM316 (WT ST-23) and BM110 (WT ST-17). Comparison of the nucleo- tide sequences of the two loci revealed that only the 5 and 3 ends of the two genes were highly conserved, displaying >90% sequence identity, whereas their internal parts displayed low-level (50–60%) or no significant (<20%) sequence identity. The positions of the prim- ers used to carry out in-frame deletion within bibA and hvgA (O1-O2 plus O3-O4), or to clone hvgA (O5-O6), are depicted by small vertical arrows. P, promoter; ter, terminator. The CovR binding site is depicted by a blue box. The pairwise local alignment was per- formed with LFasta and visualize with Laln- View (http://pbil.univ-lyon1.fr/lfasta.php). (b) Western-blot analysis of cell wall–anchored proteins of GBS with anti-HvgA antiserum. Surface proteins extracted by mutanolysin (CW) or hot SDS treatment from GBS BM110 WT ST-17, its hvgA and srtA mutants, and the complemented hvgA mutant were sepa- rated on 10% tris-glycine SDS-PAGE gels and immunoblotted with a specific anti-HvgA antiserum (protein fragment 30–216). HvgA corresponds to 2 ng of purified recombinant protein extracted from E. coli. (c) Cell surface exposure of HvgA in GBS WT ST-17. Immuno- fluorescence analysis was performed with rabbit polyclonal anti-HvgA antibodies (pro- tein fragment 30–216) revealed with an anti- IgG coupled to Alexa Fluor 488. Bars, 10 µm. (d) Flow cytometry analysis of GBS WT ST-17 and its hvgA mutant, GBS WT ST-23 and its bibA mutant incubated with a polyclonal anti-HvgA, or anti-BibA (protein fragment 34–295) antibodies and stained with a sec- ondary Alexa Fluor 488–conjugated anti– rabbit IgG antibody (solid line) or with secondary only (dotted line). (e) qRT-PCR analysis of hvgA in GBS BM110 in vitro and in vivo. Values are presented as a ratio of expression in blood, brain, and cecum of in- or no significant (<20%) sequence identity ( Fig. 2 a). We released in the cul- fected BALB/c mice relative to expression in thus investigated the contribution of Gbs2018C (hereafter ture supernatant of TH broth medium. Results shown are repre- named HvgA for hypervirulent GBS adhesin) to GBS neona- the srtA mutant, sentative of two independent experiments tal infection using in vitro and in vivo approaches, with the but not of the WT performed in triplicate. Error bars are the SD of the depicted variable. *, P < 0.05. hypothesis that it might be responsible for enhanced viru- GBS BM110. Col- lence capacities of the ST-17 clone. We first demonstrated, lectively, these re- by immunoblotting using specific anti-HvgA antibodies that sults demonstrate HvgA in GBS BM110, a prototype ST-17 strain, harbors an that HvgA is a protein anchored to the cell wall by sortase A. LPXTG motif that anchors it to the cell wall in a sortase A– Flow cytometry and immunou fl orescence microscopy con - dependent manner. As shown in Fig. 2 b, a band correspond- r fi med surface expression of HvgA in GBS WT ST-17 (Fig. 2, ing to HvgA was detected in cell wall extracts of the WT c and d). To investigate HvgA expression in vivo, quantita- strain, but not of an isogenic srtA mutant strain. Analysis of tive RT-PCR (qRT-PCR) on mRNAs extracted from the corresponding culture supernatant demonstrated that this cecal, blood, and brain samples of orally or i.v. infected mice protein is not secreted in the medium by the WT strain (see Materials and methods) were performed and demonstrated (unpublished data). Moreover, after incubation in SDS at that hvgA in vivo expression, relative to that of rpoB, is two- high temperature (10 min at 100°C), HvgA is massively to fourfold higher than in vitro (Fig. 2 e). Moreover, in total 2316 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Figure 3. HvgA promotes specific hyper- adhesion to epithelial and endothelial cells. (a and b) Comparison of the adhesive properties of ST-17 and non–ST-17 GBS strains. Cells were cultured for 1 h with bacte- ria, washed three times, and lysed, and CFUs were enumerated after plating on TH agar plates. Values are expressed as the percent- age of adhesion relative to the inoculum. (c and d) Adhesion of GBS BM110 WT (ST-17) and hvgA mutant to various cell lines, pri- mary choroid plexus epithelial cells, and brain microvessel endothelial cells. Adhesion assays were conducted as described in a and b. Streptococci were labeled with a pAb-GBS (green), nuclei were labeled with Dapi (blue) and ZO1 with an anti-ZO1 antibody, and F-actin was labeled with phalloidin (red). Bar, 20 µm. Throughout this g fi ure, results are repre - sentative of at least three independent experi- ments performed in triplicate. Error bars represent the SD. Asterisks indicate significant differences relative to BM110 WT or GBS ST-17 strains, as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). NS, a nonsignificant difference. and BibA, respectively (Table S1). Whereas both strains adhere simi- larly to A549 pulmonary epithelial cells, the ST-17 strain adheres signifi - cantly more efficiently to the intesti - nal epithelial Caco-2 cell line, the BBB-constituting cells hCMEC/D3, brain primary microvessel endothelial cells (MVECs), and choroid plexus epithelial cells (CPECs; Fig. 3 a). The significance of these results was broadened by the study of 20 randomly picked invasive GBS neonatal isolates of ST-17 type (n = 10) or non–ST-17 type (n = 10; strain characteristics de- human blood, hvgA is similarly overexpressed by threefold scribed in Table S2). As seen for the prototype strains, com- relative to standard culture medium (unpublished data). As for parative cell binding assays showed that ST-17 isolates adhere gbs2018A/bibA (Lamy et al., 2004; Mereghetti et al., 2008), significantly more to Caco-2 and hCMEC/D3 cells than hvgA transcription is up-regulated 85-fold in a 2-component non–ST-17 isolates, but not to A549 cells (Fig. 3 b), thereby regulatory system CovSR mutant (BM110covR; unpublished suggesting that bacteria expressing HvgA could display a spe- data). Together, these data show that HvgA is expressed on cific enhanced capacity to adhere to cells of the intestinal and GBS ST-17 surface and that its expression is up-regulated in blood–brain barriers. To further investigate whether HvgA is in vivo conditions. the adhesin involved in the ST-17 interaction with intestinal and blood–brain barriers constituting cells, a GBS BM110hvgA HvgA promotes specific GBS adhesion to epithelial deletion mutant was constructed (Fig. 1 a and Tables S1 and S3). and endothelial cells As expected, HvgA was not expressed in this mutant and not Because these data pointed to HvgA as a potential ST-17– detected at the bacterial cell surface (Fig. 2, b–d). The growth specific determinant conferring selective adhesive properties characteristics and the viability of the mutant in various culture to GBS, we compared the adhesion to different cell types of media (Todd-Hewitt, RPMI, or DME complemented with two reference strains, BM110 serotype III ST-17 (WT ST-17) 10% human serum), in total human blood, as well as the and NEM316 serotype III ST-23 (WT ST-23) expressing HvgA morphological characteristics and the aggregative properties JEM VOL. 207, October 25, 2010 2317 of the streptococcal chains, were similar to that of the parental intestinal barrier. To test this hypothesis, we used inbred WT strain (unpublished data). We then compared the adhe- BALB/c mice, as we observed that they displayed higher sion properties of the hvgA mutant to its isogenic parent, susceptibility than outbred Swiss mice upon oral infection. and showed that it exhibited a significantly reduced adhesion Moreover, we also observed that 4–5-wk-old mice were more to a series of epithelial and endothelial human cell lines and resistant upon oral challenge than preweaning (15–21-d-old) rodent primary cells (Fig. 3, c and d). The direct involvement BALB/c mice. We therefore inoculated orally preweaning 10 9 of HvgA in bacterial adhesion to cells was further established. (15–21-d-old) BALB/c mice with 10 or 2 × 10 CFUs. Indeed, trans-complementation with a plasmid-driving hvgA Their mortality rate 12 h after infection was signicantly fi de - expression (PhvgA) restored BM110hvgA strain adhesive creased when inoculated with the hvgA mutant as compared properties to a level similar to that of the WT strain (Fig. 4 a). to inoculation with the WT strain (Fig. 5 g). Furthermore, 8 h Furthermore, introduction of PhvgA in Lactococcus lactis enabled HvgA expression on the lactococcal surface, as assessed by im- munofluorescence microscopy and flow cytometry(Fig. 4, b and c). Trans-complemented L. lactis strains were used in assays as described for GBS to investigate HvgA-mediated adhesion: a strain expressing HvgA adhered signic fi antly more efficiently to Caco-2 and hCMEC/D3 cells than L. lactis with a vector without insert and L. lactis expressing BibA (Fig. 4 d). The nonpathogenic species L. lactis is intrinsically nonadhesive, yet heterologous expression of HvgA in L. lactis confers a clear adhesive phenotype. Moreover, a bibA mu- tant expressing HvgA adhered 10-fold more efficiently to hCMEC/D3 cells, as compared with the WT non–ST-17 (NEM316) strain (Fig. 4 e). Thus, replacement of bibA by hvgA in GBS NEM316 confers an ST-17 adhesion pheno- type to this non–ST-17 strain, and establishes the specific ad - hesive property conferred upon HvgA expression. HvgA is critical for GBS intestinal colonization and translocation across the intestinal barrier These in vitro data argued for a key contribution of HvgA in the ability of ST-17 GBS to adhere to cells constituting the intestinal and blood–brain barriers, which are targeted during LOD. We investigated this issue in the in vivo context and used mouse models of GBS infection closely mimicking the Figure 4. HvgA is necessary and sufficient to promote adhesion of pathology observed in the human neonate. We r fi st moni - GBS and L. lactis to epithelial and endothelial cells. (a) Comparison tored fecal shedding after oral inoculation (10 CFUs) of of the adhesive properties of WT ST-17 GBS and hvgA mutant or com- Swiss female mice (3–4 wk old) with WT ST-17 (BM110), its plemented strain on intestinal epithelial (Caco-2) and brain microvascular isogenic mutant hvgA or WT ST-23 (NEM316), to assess endothelial (hCMEC/D3) cell lines. Adhesion assays were conducted as described for Fig. 3, performed in triplicate, and repeated independently at their respective ability to colonize the intestine. Quantic fi a - least 3 times; graphs show cumulative data from all experiments. Error tion of GBS in feces showed that: a WT ST-17 strain estab- bars represent the SD. ***, Mann-Whitney test (P < 0.005), relative to WT lishes in the intestine more than one order of magnitude better ST-17. (b and c) Expression of HvgA in L. lactis. (b) Immunofluorescence than a non–ST-17 strain (Fig. 5 a), HvgA strongly contributes analysis was performed with rabbit polyclonal anti-HvgA antibodies (pro- to this ST-17–specic fi phenotype (Fig. 5 b), and the non–ST-17 tein fragment 30–216) revealed with an anti-IgG coupled to Alexa Fluor allelic variant BibA has no signic fi ant impact on gut col onization 488. Bars, 10 µm. (c) Flow cytometry analysis of L. lactis expressing HvgA (Fig. 5 c). Ex vivo experiments with cecal tissue explants also incubated with a polyclonal anti-HvgA antibody and stained with an demonstrated an HvgA-mediated bacterial association to the Alexa Fluor 488–conjugated anti–rabbit IgG antibody (black line histogram). (d) Comparison of adhesive properties of L. lactis and HvgA- or BibA- cecal epithelium (Fig. 5 d). In addition, competition experi- expressing strains on Caco-2 and hCMEC/D3 cell lines. Error bars repre- ments for intestinal colonization in which 5 × 10 CFUs of sent the SD. *, Mann-Whitney test (P < 0.05), relative to L. lactis + vector. each strain were simultaneously inoculated indicated that within (e) hvgA confers to WT non–ST-17 and ST-17 adhesion phenotype. Com- <1 wk, ST-17 WT totally out-competes a non–ST-17 WT parison of adhesive properties of WT non–ST-17 and bibA mutant or strain (Fig. 5 e), as well as an isogenic hvgA mutant (Fig. 5 f). complemented strains expressing BibA or HvgA on brain microvascular Together, these results demonstrate that HvgA confers a selec- endothelial cell line hCMEC/D3. Experiments were performed in triplicate tive advantage at this initial step of infection. and repeated independently at least three times; graphs show cumulative We next addressed whether HvgA could provide the data from all experiments. **, Mann-Whitney test (P < 0.01), relative to WT ST-17 strain with an enhanced ability to translocate across the non ST-17. 2318 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Figure 5. HvgA enables GBS persistent intestinal colonization and promotes its crossing of the intestinal barrier. (a–c) Groups (n = 6) of 3–4-wk-old Swiss female mice were infected orally with 10 CFUs WT GBS or mutant strains, and fecal shedding was assessed 3, 7, and 14 d after inoculation by CFUs enumeration. Values represent fecal shedding of the six mice in a cage. (d) Infec- tion of Swiss mice (n = 4) ligated cecum with 10 GBS WT ST-17 or hvgA mutant strain. Bacteria were recovered 1 h after infection. Values are expressed as the percentage of adhesion relative to the inoculum. (e and f) Competition assays between WT ST-17 (BM110) and WT non–ST-17 (NEM316) or hvgA BM110 mutant. 3–4-wk-old Swiss mice (n = 6) were infected orally with a 1:1 mixture of the two strains (total dose 10 CFUs). Bacteria were enumerated from the feces collected 3, 7, and 10 d after infection. (g) Mortality rate 12 h after infection of 15–21 d old BALB/c mice (n = 10) infected orally with the WT ST-17 or hvgA mutant strains. (h) Groups of 4-wk-old BALB/c mice (n = 10) were inoculated orally with 10 CFUs WT ST-17 or hvgA mutant strains. 8 h after infection, animals were sacrificed. Cecum were collected and divided longitudinally in two parts. One half was directly homogenized: extra- and intratissular bacteria associated with the cecum were enumerated (no genta.). The second half was incubated for 3 h in DMEM containing 250 µg/ml gentamicin to kill extratissular bacteria and homogenized. Intratissular invading bacteria were then enumerated (genta.). Animal experiments represented in this figure were repeated at least two times and groups of mice contained at least five animals. Error bars represent the SD of depicted variable performed in triplicate. Asterisks indicate significant differences as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). after oral inoculation of 4–5-wk-old BALB/c mice with 10 microscopy (Fig. S2). In mice with the highest WT-ST17 bac- 9 7 or 10 , no mortality was observed. But here again, the WT terial load (5 × 10 CFUs) in the brain, GBS were observed ST-17 strain adhered to and invaded the cecal tissue more adhering to the inner meningeal envelopes (pia matter, Fig. S2 a) significantly than the hvgA mutant (Fig. 5 h and Fig. S1 a), in the brain microvessel (Fig. S2 b), in the brain parenchyma indicating a key role for HvgA in GBS ST-17 ability to cross (Fig. S2 c), or in the choroid plexuses (Fig. S2 d). In contrast, the intestinal barrier. Moreover, in germ-free animals infected no bacterium was observed in mice infected with the hvgA with WT GBS ST-17, bacteria could be observed adhering mutant. To focus specifically on the crossing of the BBB and to the enterocytes and in the lamina propria (Fig. S1, b-e), bypass the HvgA phenotype at the BALB/c intestinal barrier similar to what was observed in the human previously de- level, we developed a CNS infection model in which pre- scribed LOD case (Fig. 1, a and b) weaning 3 wk mice were infected i.v. every 12 h with a rela- tively low inoculum (5 × 10 CFUs) to avoid unspecific BBB HvgA contributes to GBS crossing of the blood–brain barrier opening caused by the massive systemic inflammation trig - and the onset of meningitis gered by high bacterial loads, but to instead mimic blood- We next analyzed HvgA contribution to central nervous system borne neonatal meningitis. Monitoring of bacterial loads in (CNS) invasion. A role for HvgA in this process was supported the blood and the CNS showed that whereas WT ST-17 and by results obtained with orally inoculated animals: 15–21-d-old its hvgA isogenic mutant induce a similar level of bacteremia BALB/c mice infected with the WT GBS-ST17 strain have 48 h after i.v. inoculation (Fig. 6 c), hvgA is significantly im - significantly higher bacterial counts in the brain than the paired in its ability to invade the CNS (Fig. 6 d). In agreement mice infected with the hvgA mutant at the experiment end- with these quantitative data, real-time imaging of biolumines- point (Fig. 6, a and b). Moreover, animals that died in the cent bacteria along the infectious process revealed a marked meantime are those for which bacterial counts in the brain bioluminescence emission in the head of animals infected are the highest (Fig. 6 a), arguing for a causal relationship with WT ST-17 (4/4 animals), which was detectable 20 h after between HvgA expression and GBS ST-17 ability to reach infection and is visible until the death of the animal, whereas the CNS after oral inoculation. To investigate the onset of merely no signal was detected in mice infected with the meningitis in these orally inoculated animals, their brains hvgA mutant (Fig. 6 e). Neuropathological analysis of in- were examined by immunohistochemistry and confocal fected animals 48 h after i.v. inoculation disclosed an intense JEM VOL. 207, October 25, 2010 2319 Figure 6. HvgA enables GBS crossing of the BBBs. (a and b) Groups of 15–21 d old BALB/c mice (n > 8) 9 8 were inoculated orally with 10 (a) or 5 × 10 CFUs (b) WT ST-17 or hvgA mutant strain and brain invasion was assessed 12-h after inoculation by CFUs enumeration. (c and d) Groups of 3–4-wk-old BALB/c mice (n > 8) were infected by repeated i.v. injections of 5 × 10 CFUs every 12 h and sacrificed at 48 h, and bacteria enumerated in blood (c) and brain (d). Data shown are the results of three independent experiments. (e) Real-time imaging of CNS infection. Groups of BALB/c mice (n = 4) were in- fected i.v. with 10 CFUs every 12 h. Bioluminescence was assessed every 12 h (the images shown correspond to the 36 h after injection time point). (f–h) Histopathologi- cal examination (Gram staining) of CNS tissue samples from WT-ST-17 infected mice reveals Gram-positive cocci in the meninges (f), in the choroid plexuses (g), in brain microvessel endothelial cells and the surrounding brain parenchyma (h). (i–k) Immunofluorescence staining of brain sections obtained from animals infected with the WT ST-17 in d, showing infection of meninges (i), sub- meningeal space (j), and brain microvessel endothelial cells (k). Bars: (f–h) 25 µm; (i–5) 20 µm. All experiments were repeated at least three times. NS, not significant. Asterisks indicate significant differences as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). associated with ST-17 GBS isolates: (1) their close association with LOD, which can now be linked to HvgA-mediated intestinal colonization and subsequent crossing of the intestinal barrier, and (2) their close association with meningitis, which can now be linked to the HvgA-mediated cross- ing of the BBB. This study den fi itely establishes the hypervirulence of the ST-17 GBS clone and links it to enhanced intestinal colonization and barrier breaching potentials. Importantly, it illustrates how a microbial factor implicated in bacterial intestinal colonization can behave as a specific virulence factor in the neonatal con - text, at a time when the intestinal microflora is not yet established and cannot exerts its buffer - WT ST-17 streptococcal infection of choroid plexuses and ing effect on potential pathogenic bacteria such as GBS. Given meninges (Fig. 6, f, g, i, and k), as well as in brain microvessels the burden of ST-17–associated LOD and neonatal meningi- and the surrounding brain parenchyma (Fig. 6, h and k), a result tis (Poyart et al., 2008; CDC, 2009), hvgA and its gene product are matching our observations in the human LOD case associated now promising targets for developing diagnostic tools (Lamy with ST-17 GBS (Fig. 1, c–l). Together, these in vivo results dem- et al., 2006) and vaccines (Santi et al., 2009), respectively. onstrate HvgA contribution in ST-17 access to the CNS, a result that corroborates our in vitro data (Fig. 3, c and d). MATERIAL AND METHODS Bacterial strains, genetic constructions, and growth conditions. The Conclusions main characteristics of bacterial strains and plasmids used in this study are listed in Table S1. GBS NEM316, capsular serotype III, and MLST type ST Using complementary approaches, we have uncovered a key 23, and GBS BM110, capsular serotype III, and MLST sequence type ST-17 determinant of the pathophysiology of ST-17–associated are well-characterized isolates from human with invasive infections. GBS LOD. We have identified an ST-17–specific surface-expressed clinical strains from invasive infection were collected by the National Refer- protein, HvgA, and demonstrated that it is a major determi- ence Center for Streptococci from 2006 and 2009. GBS mutants were nant of ST-17 hypervirulence in neonates. Our results pro- constructed by in-frame deletions in bibA (GBS NEM316), hvgA (GBS vide a rational explanation for the two main characteristics BM110), and srtA (GBS BM110), as previously described (Dramsi et al., 2006) 2320 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t using primers listed in Table S3 and Fig. 2 a. The expected in-frame dele- injected intragastrically. At the indicated times, cecum were collected, rinsed tions were confirmed by PCR and sequence analysis. Complemented GBS in DME, and divided in two parts longitudinally. One half was directly ho- mutant strains and L. lactis expressing BibA or HvgA were constructed as mogenized in PBS with a tissue homogenizer. The second half was incu- follows. bibA and hvgA genes were amplified by PCR from chromosomal bated for 3 h in DME containing 250 µg/ml gentamicin to determine DNA of GBS NEM316 and BM110, respectively, using primers O5 and O6 bacterial invasion, rinsed, and homogenized. Serial dilutions were plated on (Fig. 2 a and Table S3) and cloned into the pOri23 shuttle vector. Recombi- selective medium (Granada) for CFU counts. For cecum-ligated loop ex- nant plasmids PbibA and PhvgA were introduced by electroporation into periments, animals were anesthetized. The whole cecums were closed at GBS mutants and L. lactis. Unless otherwise specified, GBS and L. lactis were both ends with surgical sutures. 200 µl of the adequate bacterial dilution was cultured at 37°C in Todd Hewitt (TH) broth or agar and antibiotics were injected in the lumen. 1 h after infection, the mice were sacric fi ed; the cecum- used at the following concentrations: erythromycin, 10 µg.ml ; kanamycin, ligated loops were excised and opened longitudinally. For competitive assay, 1,000 µg.ml . both WT and isogenic mutants were mixed in a 1:1 ratio oral infection ex- periments. At the indicated times, stools were collected and serial dilutions Generation of anti-BibA and anti-HvgA rabbit polyclonal anti- were plated on selective medium. i.v. infections were performed by i.v. in- bodies. Recombinant Gbs2018 segments were expressed and purie fi d as fol - jection every 12 h for 36 h. All of the procedures were in agreement with lows. DNA fragments intragenic to bibA (nt 100–1185) and hvgA (nt 88–648) the guidelines of the European Commission for the handling of laboratory were produced by PCR using genomic DNA of GBS NEM316 and BM110, animals, directive 86/609/EEC (http://ec.europa.eu/environment/chemicals/ respectively, as templates, as well as the primers listed in Table S3. DNA frag- lab_animals/home_en.htm) and were approved by the Animal Care and Use ments were digested with the appropriate enzymes and cloned into pET- Committees of the Institut Pasteur. 26b(+) and pET2817, respectively. The resulting plasmids were introduced into E. coli BL21DE3/pDIA17 for protein expression. Recombinant proteins Bioluminescence real-time imaging. GBS WT-ST17 strain and the were purie fi d under native conditions on Ni-NTA columns (QIAGEN), fol - hvgA isogenic mutant were made bioluminescent after introduction plas- lowed by Q–Sepharose anion exchange chromatography (GE Healthcare). mid pTCVlux by electroporation (Table S1). After i.v. infection, BALB/c The purie fi d BibA or HvgA truncated proteins were injected into rabbit to mice bioluminescence was monitored each day, as previously described produce antibodies, as previously described (Lalioui et al., 2005). (Disson et al., 2009). 12 h before infection and every 12 h during the infec- tion, animals were treated with erythromycin (20 mg/kg ). RNA isolation, reverse transcription, and qRT-PCR. Total RNAs were extracted from bacteria as previously described (Lamy et al., 2004). For Tissue labeling. Thin sections, thick sections, and whole-mount tissue la- isolation of bacterial RNA from human blood, bacteria were grown in TH beling were performed as previously described (Disson et al., 2009). Acquisi- broth to the mid-logarithmic phase (OD 0.3–0.4), washed in PBS, and tion of images was realized with an upright confocal microscope (Carl Zeiss, 600nm resuspended in an equal volume of freshly drawn human blood from non- Inc.), using a 40× water immersion objective for the whole-mount tissue or immune healthy donors and incubated for 1 h at 37°C. RNA isolation from an oil immersion objective for the thick section labeling. Reconstructions animal blood and tissues was as previously described (Oggioni et al., 2006). were realized using Imaris software. cDNA synthesis was performed on DNase-I–treated RNA (50 ng of two Online supplemental material. Fig. S1 shows the role of HvgA in the independent extracts) with specific reverse primers using SuperScript II re - crossing of the intestinal barrier. Fig. S2 shows that GBS ST-17 crosses the verse transcription (Invitrogen). qRT-PCR analysis was performed as previ- BBB upon oral inoculation. Table S1 describes the bacterial strains and plas- ously described (Yamamoto et al., 2005) using primers listed in Table S3. 2Ct mids used in this study. Table S2 describes the origin, the serotype, and the The relative fold change of each gene was calculated from 2 by using ST of GBS strains used in this study. Table S3 shows the list of primers used rpoB as an internal control gene. for genetic constructions and qRT-PCR. Online supplemental material is Immunoblots and bacterial immunofluorescence stainings. For anal- available at http://www.jem.org/cgi/content/full/jem.20092594/DC1. ysis of HvgA expression, cell wall proteins were extracted as previously de- scribed (Dramsi et al., 2006, Lalioui et al., 2005). After SDS-PAGE, proteins We are grateful to Alexandra Gruss for helpful discussion and critical reading of the were transferred to nitrocellulose membrane. HvgA was detected using manuscript. We thank Pierre-Olivier Couraud for providing HCMEC/D3 cells. This work was supported by the Agence Nationale de la Recherche (ANR rabbit-specific pAb and horseradish peroxidase–coupled anti–rabbit second - HyperVirGBS Project; ANR-08-MIE-015), Institut National de la Santé et de la ary antibodies and the ECL Reagent (GE Healthcare). Immunofluorescence Recherche Médicale, Institut Pasteur, Fondation pour la Recherche Médicale (FRM), staining and fluorescence-activated cell sorter analysis were carried out as Université Paris Descartes, the Institut de Veille Sanitaire, Programme Transversal de previously described (Dramsi et al., 2006). Recherche #190. S. Bellais was a recipient of a post-doctoral fellowship from the ANR-08-MIE-015 grant and O. Disson from FRM and Institut National de la Santé Cell cultures assays. Human epithelial (Caco-2, A549, and HeLa), and et de la Recherche Médicale. human endothelial (HUVEC) cell lines are from the American Type Culture The authors declare that they have no competing financial interest. Collection. Human brain endothelial cell line hCMEC/D3 (Weksler et al., 2005) was provided by P.O. Couraud (Institut National de la Santé et de la Submitted: 4 December 2009 Recherche Médicale, 75014 Paris, France). 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The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates

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Abstract

B r i e f D e f i n i t ive R e p o r t The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates 1,2,3 4,5 1,2 Asmaa Tazi, Olivier Disson, Samuel Bellais, 1,2 3 6 Abdelouhab Bouaboud, Nicolas Dmytruk, Shaynoor Dramsi, 6 7 8 Michel-Yves Mistou, Huot Khun, Charlotte Mechler, 1,2 6 4,5,9 Isabelle Tardieux, Patrick Trieu-Cuot, Marc Lecuit, 1,2,3,6 and Claire Poyart Institut Cochin, Université Paris Descartes Faculté de Médecine, Centre National de la Recherche Scientifique (UMR 8104), 75014 Paris, France Institut National de la Santé et de la Recherche Médicale, U1016, 75014 Paris, France Assistance Publique Hôpitaux de Paris, Service de Bactériologie, Centre National de Référence des Streptocoques, Hôpital Cochin, 75014 Paris, France Institut Pasteur, Groupe Microorganismes et Barrières de l’Hôte, 75015 Paris, France Institut National de la Santé et de la Recherche Médicale Avenir U604, 75015 Paris, France Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram Positif, URA Centre National de la Recherche Scientifique 2172, 75015 Paris, France Institut Pasteur, Unité d’Histotechnologie et Pathologie, 75015 Paris, France Assistance Publique Hôpitaux de Paris, Service d’Anatomie Pathologique, Hôpital Louis Mourier, 92700 Colombes, France Université Paris Descartes Faculté de Médecine, Assistance Publique-Hôpitaux de Paris, Service des Maladies Infectieuses et Tropicales, Hôpital Necker-Enfants Malades, 75015 Paris, France Streptococcus agalactiae (group B streptococcus; GBS) is a normal constituent of the intestinal microflora and the major cause of human neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-onset disease (LOD), which is characterized by meningitis in infants after the first week of life. The pathophysiology of LOD remains poorly understood, but our epidemiological and histo- pathological results point to an oral route of infection. Here, we identify a novel ST-17– specific surface-anchored protein that we call hypervirulent GBS adhesin (HvgA), and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered more efficiently to intestinal epithelial cells, choroid plexus epithelial cells, and microvascular endothelial cells that constitute the blood–brain barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in nonadhesive bacteria conferred the ability to adhere to intestinal barrier and BBB-constituting cells. In orally inoculated mice, HvgA was required for intestinal colonization and translocation across the intestinal barrier and the BBB, leading to meningitis. In conclusion, HvgA is a critical virulence trait of GBS in the neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies. CORRESPONDENCE Claire Poyart: [email protected] Group B streptococcus (GBS; Streptococcus agalac- improvement in neonatal intensive care, up to OR tiae) is a Gram-positive encapsulated commensal 10% of neonatal GBS infections are lethal, and Marc Lecuit: bacterium of the human intestine that is also pres- 25–35% of surviving infants with meningitis [email protected] ent in the vagina of 15–30% of healthy women. experience permanent neurological sequelae Abbreviations used: BBB, blood– In neonates, it may turn into a deadly pathogen, (Edwards and Baker, 2005). GBS is also a brain barrier; CNS, central and it is the leading cause of neonatal pneumonia, nervous system; EOD, early-onset septicaemia, and meningitis (Edwards and Baker, disease; GBS, group B strepto- © 2010 Tazi et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after coccus; HvgA, hypervirulent GBS 2005). Despite early antimicrobial treatment and the publication date (see http://www.rupress.org/terms). After six months it is adhesin; LOD, late-onset disease; available under a Creative Commons License (Attribution–Noncommercial–Share qRT-PCR, quantitative RT-PCR; Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ TH, Todd Hewitt. M. Lecuit and C. Poyart contributed equally to this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 207 No. 11 2313-2322 www.jem.org/cgi/doi/10.1084/jem.20092594 The Journal of Experimental Medicine significant cause of morbidity and mortality in nonpregnant We show that this protein which we have called hypervirulent adults, particularly those with underlying diseases and the GBS adhesin (HvgA) mediates GBS neonatal intestinal coloniz- elderly (Phares et al., 2008). ation and crossing of the intestinal and blood–brain barriers, Two distinct GBS-associated clinical syndromes, referred leading to meningitis, which are key features of LOD. to as early-onset disease (EOD) and late-onset disease (LOD) have been recognized in neonates in their r fi st week of life (age RESULTS AND DISCUSSION 0–6 d) and after (age 7–89 d), respectively (Edwards and Baker, Epidemiological evidence that the ST-17 hypervirulent GBS 2005). Although intrapartum antibioprophylaxis for parturient clone is associated with LOD and neonatal meningitis women at risk for GBS infection has markedly decreased the We first analyzed 651 GBS isolates referred to the French na - incidence of EOD, it did not change that of LOD (Poyart et al., tional reference center for streptococci between 2006 and 2008; CDC, 2009). Epidemiological data collected worldwide 2009 from consecutive cases of invasive infection in neonates have shown that a substantial proportion of EOD and the ma- (meningitis, n = 138; bacteremia, n = 166) and in adults (men- jority of LOD are associated with capsular serotype III (Lin et al., ingitis, n = 16; bacteremia, n = 331). Serotype III accounts for 2006; Gherardi et al., 2007; Phares et al., 2008; Poyart et al., 86.2% of strains isolated from cases of neonatal meningitis 2008; CDC, 2009). Strains of serotype III contain a limited and 60.8% of neonatal bacteremia, but only 25.7% of bacte- number of clonal complexes, den fi ed by multilocus sequence remia in adults (Table I). Serotype III is significantly associ - typing. Among them, the ST-17 sequence type is strongly associ- ated with meningitis during EOD (79.3%; P < 0.0001) and ated with neonatal meningitis and was therefore designated as LOD (88%; P < 0.0001; Table I). Moreover, the serotype III “the hypervirulent clone,” despite the absence of experimental ST-17 clone is significantly associated with meningitis during data to support this assertion (Musser et al., 1989; Jones et al., EOD (79.3%; P < 0.0001) and LOD (82.6%; P < 0.0001), 2003, 2006; Brochet et al., 2006; Lamy et al., 2006; Bohnsack and with bacteremia during LOD (78.1%; P < 0.0001; Table I). et al., 2008; Poyart et al., 2008; Manning et al., 2009). In contrast, the ST-17 clone represents <12% of isolates from For EOD, the mode of transmission in newborns is thought adult patients with bacteremia (Table I). Together, these results to be vertical, by inhalation of GBS-contaminated amniotic or obtained from a total of 651 clinical strains demonstrate that vaginal fluid during parturition, followed by bacterial trans - ST-17 GBS strains account for >80% of neonatal meningitis, location across the respiratory epithelium and subsequent sys- strongly suggesting an enhanced virulence of the ST-17 clonal temic infection (Edwards and Baker, 2005). In contrast, for complex in the neonatal context. These epidemiological ob- LOD, the mode of transmission and the infection route remain servations thus prompted us to search for specific virulence elusive, although mother-to-child transmission might also be factors of the ST-17 clone that may account for its apparent involved. A plausible scenario would involve early intestinal higher pathogenicity in neonates, its close association with colonization by GBS that would lead in the r fi st days of life to LOD, and its meningeal tropism. its intraluminal intestinal multiplication, translocation across the intestinal epithelium, and access to the bloodstream. Histopathological study of a fatal case Indeed, an intestinal portal of entry for LOD is supported by of ST-17–associated LOD several lines of evidence: (a) 60 and 40% of the neonates asymp- A term female infant (gestational age, 39 wk; birth weight, tomatically colonized with GBS at birth remain positive for 3,140 g) was born by spontaneous vaginal delivery without bacteria at the rectal level at 4 and 12 wk of life, respectively complication. Maternal vaginal swab at 37 wk of gestation (Weindling et al., 1981); and (b) a longitudinal study of GBS was negative for GBS. There was no premature membrane vaginal and rectal colonization in women during and after rupture and neither skin nor rectal swab of the neonate was pregnancy has revealed that carriers are usually colonized for made at delivery. The mother and her breastfed baby were up to 2 yr by a single clone, which is also frequently found in discharged on day 4. On day 14 of life, the neonate developed newborn feces for up to 1 yr (Hansen et al., 2004). muscular hypotonia, poor suckling, hyperexcitability, and Once translocated in the bloodstream, GBS has the ability fever. Cerebrospinal fluid and blood cultures were positive for to cross the blood–brain barrier (BBB) and cause meningitis. GBS, which was later shown to belong to serotype III and Several virulence factors contribute to the pathogenesis of GBS clonal complex ST-17. Breast milk was not cultivated. Despite meningitis in animal models, but nearly all of them are in- adequate antimicrobial treatment associating amoxicillin, cef- volved in the septicemia phase of the infection, but not in GBS triaxone, and gentamicin, she died 8 h later and an autopsy adhesion to and crossing of the BBB (Maisey et al., 2008). One was performed. Cultures of stool, blood, and cerebrospinal fluid, exception is Srr-1, a recently characterized surface glycoprotein as well as colonic and brain autopsic tissue samples, were all that promotes adhesion to and invasion of human brain micro- positive for GBS. Immunohistochemistry of paran ffi -embedded vascular endothelial cells and contributes to BBB crossing in gut tissue samples led to the detection of GBS associated with mice (van Sorge et al., 2009). This illustrates that more studies the intestinal tissue and inside the lamina propria (Fig. 1, a and b). are needed to identify virulence factors of GBS, especially in GBS also heavily infected meningeal tissues, with intense in- regard to its meningeal tropism and its ability to trigger LOD. flammation indicated by the massive recruitment of poly - Here, we have identie fi d a novel ST-17–specic fi surface- morphonuclear cells (Fig. 1, c and d). GBS was also observed anchored protein, which is highly prevalent in cases of LOD. to be tightly associated with brain microvessel endothelial 2314 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Table I. Serotype and ST-17 distribution of 651 GBS strains isolated from neonatal and non-pregnant adult invasive infections in France between 2006 and 2009 Type of infection Serotype no. ST-17 no. Ia Ib II III IV V Total % % Neonatal meningitis 12 (8.7) 4 (2.9) 0 119 (86.2) 0 3 (2.1) 138 (100) 113 (81.9) EOD (≤6 d) 6 (20.7) 0 0 23 (79.3) 0 0 29 (100) 23 (79.3) LOD (≥7–89 d) 6 (5.5) 4 (3.7) 0 96 (88.1) 0 3 (2.7) 109 (100) 90 (82.6) Neonatal bacteremia 34 (20.5) 5 (3) 6 (3.6) 101 (60.8) 3 (1.8) 17 (10.2) 166 (100) 89 (53.6) EOD (≤6 d) 27 (29) 3 (3.2) 5 (5.4) 41 (44.1) 2 (2.2) 15 (16.1) 93 (100) 32 (34.4) LOD (≥7–89 d) 7 (9.6) 2 (2.7) 1 (1.4) 60 (82.2) 1 (1.4) 2 (2.7) 73 (100) 57 (78.1) Adult bacteremia 71 (21.4) 36 (10.3) 37 (11.2) 85 (25.7) 17 (5.1) 85 (25.7) 331 (100) 37 (11.2) Adult meningitis 4 (25) 2 (12.5) 1 (6.25) 8 (50) 0 1 (6.25) 16 (100) 5 (31.3) cells and choroid plexus epithelial cells, which constitute the HvgA is an ST-17–specific surface-anchored protein blood–brain parenchyma and blood–cerebrospinal fluid bar - that is overexpressed in vivo riers, respectively (Fig. 1, e–l). These bacteriological and histo- We first analyzed whether the ST-17 clone expresses specific pathological analyses of this fatal case of LOD are consistent surface-exposed molecules that could account for enhanced with the hypothesis that LOD results from the ability of GBS adhesive properties. The comparative analysis of GBS whole- ST-17 to efficiently colonize the intestine, cross the intestinal genome sequences has pinpointed several genes encoding surface components specic fi to the ST-17 clone (Tettelin et al., barrier, and cross the BBB. 2005; Brochet et al., 2006). In partic- ular, we have identie fi d mosaic variants at a single genomic locus (Lamy et al., 2006) encoding a cell wall–anchored protein, with two main variants dis- playing 38% overall amino acid iden- tity, namely Gbs2018A, which is also referred to as BibA (Santi et al., 2007), and Gbs2018C, which we have shown to be strictly specific to the “hyper- virulent” ST-17 clone (Lamy et al., 2006). These genes have conserved reg- ulatory regions and encode proteins with conserved N- and C-terminal parts, but a distinct central core. Indeed, comparison of the nucleotide sequences of the two loci has revealed that only the 5 and 3 ends of the two genes are highly conserved, displaying >90% sequence identity, whereas their in- ternal parts display low level (50–60%) Figure 1. ST-17 GBS crossing of the intestinal and BBBs in a fatal case of human neonatal LOD with meningitis. Immunohistological study of the intestine and the CNS of a fatal case of ST-17 LOD. Bacteria were labeled with a specific polyclonal anti - body to GBS and appear in reddish brown. Sections were counterstained with hematoxy- lin. GBS is present in the intestine (a and b), in meninges (c and d), in brain microvessels (e–j), as well as in choroid plexuses (k and l). JEM VOL. 207, October 25, 2010 2315 Figure 2. HvgA is a cell surface protein of GBS ST-17. (a) Structure of the bibA/hvgA locus in GBS strains NEM316 (WT ST-23) and BM110 (WT ST-17). Comparison of the nucleo- tide sequences of the two loci revealed that only the 5 and 3 ends of the two genes were highly conserved, displaying >90% sequence identity, whereas their internal parts displayed low-level (50–60%) or no significant (<20%) sequence identity. The positions of the prim- ers used to carry out in-frame deletion within bibA and hvgA (O1-O2 plus O3-O4), or to clone hvgA (O5-O6), are depicted by small vertical arrows. P, promoter; ter, terminator. The CovR binding site is depicted by a blue box. The pairwise local alignment was per- formed with LFasta and visualize with Laln- View (http://pbil.univ-lyon1.fr/lfasta.php). (b) Western-blot analysis of cell wall–anchored proteins of GBS with anti-HvgA antiserum. Surface proteins extracted by mutanolysin (CW) or hot SDS treatment from GBS BM110 WT ST-17, its hvgA and srtA mutants, and the complemented hvgA mutant were sepa- rated on 10% tris-glycine SDS-PAGE gels and immunoblotted with a specific anti-HvgA antiserum (protein fragment 30–216). HvgA corresponds to 2 ng of purified recombinant protein extracted from E. coli. (c) Cell surface exposure of HvgA in GBS WT ST-17. Immuno- fluorescence analysis was performed with rabbit polyclonal anti-HvgA antibodies (pro- tein fragment 30–216) revealed with an anti- IgG coupled to Alexa Fluor 488. Bars, 10 µm. (d) Flow cytometry analysis of GBS WT ST-17 and its hvgA mutant, GBS WT ST-23 and its bibA mutant incubated with a polyclonal anti-HvgA, or anti-BibA (protein fragment 34–295) antibodies and stained with a sec- ondary Alexa Fluor 488–conjugated anti– rabbit IgG antibody (solid line) or with secondary only (dotted line). (e) qRT-PCR analysis of hvgA in GBS BM110 in vitro and in vivo. Values are presented as a ratio of expression in blood, brain, and cecum of in- or no significant (<20%) sequence identity ( Fig. 2 a). We released in the cul- fected BALB/c mice relative to expression in thus investigated the contribution of Gbs2018C (hereafter ture supernatant of TH broth medium. Results shown are repre- named HvgA for hypervirulent GBS adhesin) to GBS neona- the srtA mutant, sentative of two independent experiments tal infection using in vitro and in vivo approaches, with the but not of the WT performed in triplicate. Error bars are the SD of the depicted variable. *, P < 0.05. hypothesis that it might be responsible for enhanced viru- GBS BM110. Col- lence capacities of the ST-17 clone. We first demonstrated, lectively, these re- by immunoblotting using specific anti-HvgA antibodies that sults demonstrate HvgA in GBS BM110, a prototype ST-17 strain, harbors an that HvgA is a protein anchored to the cell wall by sortase A. LPXTG motif that anchors it to the cell wall in a sortase A– Flow cytometry and immunou fl orescence microscopy con - dependent manner. As shown in Fig. 2 b, a band correspond- r fi med surface expression of HvgA in GBS WT ST-17 (Fig. 2, ing to HvgA was detected in cell wall extracts of the WT c and d). To investigate HvgA expression in vivo, quantita- strain, but not of an isogenic srtA mutant strain. Analysis of tive RT-PCR (qRT-PCR) on mRNAs extracted from the corresponding culture supernatant demonstrated that this cecal, blood, and brain samples of orally or i.v. infected mice protein is not secreted in the medium by the WT strain (see Materials and methods) were performed and demonstrated (unpublished data). Moreover, after incubation in SDS at that hvgA in vivo expression, relative to that of rpoB, is two- high temperature (10 min at 100°C), HvgA is massively to fourfold higher than in vitro (Fig. 2 e). Moreover, in total 2316 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Figure 3. HvgA promotes specific hyper- adhesion to epithelial and endothelial cells. (a and b) Comparison of the adhesive properties of ST-17 and non–ST-17 GBS strains. Cells were cultured for 1 h with bacte- ria, washed three times, and lysed, and CFUs were enumerated after plating on TH agar plates. Values are expressed as the percent- age of adhesion relative to the inoculum. (c and d) Adhesion of GBS BM110 WT (ST-17) and hvgA mutant to various cell lines, pri- mary choroid plexus epithelial cells, and brain microvessel endothelial cells. Adhesion assays were conducted as described in a and b. Streptococci were labeled with a pAb-GBS (green), nuclei were labeled with Dapi (blue) and ZO1 with an anti-ZO1 antibody, and F-actin was labeled with phalloidin (red). Bar, 20 µm. Throughout this g fi ure, results are repre - sentative of at least three independent experi- ments performed in triplicate. Error bars represent the SD. Asterisks indicate significant differences relative to BM110 WT or GBS ST-17 strains, as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). NS, a nonsignificant difference. and BibA, respectively (Table S1). Whereas both strains adhere simi- larly to A549 pulmonary epithelial cells, the ST-17 strain adheres signifi - cantly more efficiently to the intesti - nal epithelial Caco-2 cell line, the BBB-constituting cells hCMEC/D3, brain primary microvessel endothelial cells (MVECs), and choroid plexus epithelial cells (CPECs; Fig. 3 a). The significance of these results was broadened by the study of 20 randomly picked invasive GBS neonatal isolates of ST-17 type (n = 10) or non–ST-17 type (n = 10; strain characteristics de- human blood, hvgA is similarly overexpressed by threefold scribed in Table S2). As seen for the prototype strains, com- relative to standard culture medium (unpublished data). As for parative cell binding assays showed that ST-17 isolates adhere gbs2018A/bibA (Lamy et al., 2004; Mereghetti et al., 2008), significantly more to Caco-2 and hCMEC/D3 cells than hvgA transcription is up-regulated 85-fold in a 2-component non–ST-17 isolates, but not to A549 cells (Fig. 3 b), thereby regulatory system CovSR mutant (BM110covR; unpublished suggesting that bacteria expressing HvgA could display a spe- data). Together, these data show that HvgA is expressed on cific enhanced capacity to adhere to cells of the intestinal and GBS ST-17 surface and that its expression is up-regulated in blood–brain barriers. To further investigate whether HvgA is in vivo conditions. the adhesin involved in the ST-17 interaction with intestinal and blood–brain barriers constituting cells, a GBS BM110hvgA HvgA promotes specific GBS adhesion to epithelial deletion mutant was constructed (Fig. 1 a and Tables S1 and S3). and endothelial cells As expected, HvgA was not expressed in this mutant and not Because these data pointed to HvgA as a potential ST-17– detected at the bacterial cell surface (Fig. 2, b–d). The growth specific determinant conferring selective adhesive properties characteristics and the viability of the mutant in various culture to GBS, we compared the adhesion to different cell types of media (Todd-Hewitt, RPMI, or DME complemented with two reference strains, BM110 serotype III ST-17 (WT ST-17) 10% human serum), in total human blood, as well as the and NEM316 serotype III ST-23 (WT ST-23) expressing HvgA morphological characteristics and the aggregative properties JEM VOL. 207, October 25, 2010 2317 of the streptococcal chains, were similar to that of the parental intestinal barrier. To test this hypothesis, we used inbred WT strain (unpublished data). We then compared the adhe- BALB/c mice, as we observed that they displayed higher sion properties of the hvgA mutant to its isogenic parent, susceptibility than outbred Swiss mice upon oral infection. and showed that it exhibited a significantly reduced adhesion Moreover, we also observed that 4–5-wk-old mice were more to a series of epithelial and endothelial human cell lines and resistant upon oral challenge than preweaning (15–21-d-old) rodent primary cells (Fig. 3, c and d). The direct involvement BALB/c mice. We therefore inoculated orally preweaning 10 9 of HvgA in bacterial adhesion to cells was further established. (15–21-d-old) BALB/c mice with 10 or 2 × 10 CFUs. Indeed, trans-complementation with a plasmid-driving hvgA Their mortality rate 12 h after infection was signicantly fi de - expression (PhvgA) restored BM110hvgA strain adhesive creased when inoculated with the hvgA mutant as compared properties to a level similar to that of the WT strain (Fig. 4 a). to inoculation with the WT strain (Fig. 5 g). Furthermore, 8 h Furthermore, introduction of PhvgA in Lactococcus lactis enabled HvgA expression on the lactococcal surface, as assessed by im- munofluorescence microscopy and flow cytometry(Fig. 4, b and c). Trans-complemented L. lactis strains were used in assays as described for GBS to investigate HvgA-mediated adhesion: a strain expressing HvgA adhered signic fi antly more efficiently to Caco-2 and hCMEC/D3 cells than L. lactis with a vector without insert and L. lactis expressing BibA (Fig. 4 d). The nonpathogenic species L. lactis is intrinsically nonadhesive, yet heterologous expression of HvgA in L. lactis confers a clear adhesive phenotype. Moreover, a bibA mu- tant expressing HvgA adhered 10-fold more efficiently to hCMEC/D3 cells, as compared with the WT non–ST-17 (NEM316) strain (Fig. 4 e). Thus, replacement of bibA by hvgA in GBS NEM316 confers an ST-17 adhesion pheno- type to this non–ST-17 strain, and establishes the specific ad - hesive property conferred upon HvgA expression. HvgA is critical for GBS intestinal colonization and translocation across the intestinal barrier These in vitro data argued for a key contribution of HvgA in the ability of ST-17 GBS to adhere to cells constituting the intestinal and blood–brain barriers, which are targeted during LOD. We investigated this issue in the in vivo context and used mouse models of GBS infection closely mimicking the Figure 4. HvgA is necessary and sufficient to promote adhesion of pathology observed in the human neonate. We r fi st moni - GBS and L. lactis to epithelial and endothelial cells. (a) Comparison tored fecal shedding after oral inoculation (10 CFUs) of of the adhesive properties of WT ST-17 GBS and hvgA mutant or com- Swiss female mice (3–4 wk old) with WT ST-17 (BM110), its plemented strain on intestinal epithelial (Caco-2) and brain microvascular isogenic mutant hvgA or WT ST-23 (NEM316), to assess endothelial (hCMEC/D3) cell lines. Adhesion assays were conducted as described for Fig. 3, performed in triplicate, and repeated independently at their respective ability to colonize the intestine. Quantic fi a - least 3 times; graphs show cumulative data from all experiments. Error tion of GBS in feces showed that: a WT ST-17 strain estab- bars represent the SD. ***, Mann-Whitney test (P < 0.005), relative to WT lishes in the intestine more than one order of magnitude better ST-17. (b and c) Expression of HvgA in L. lactis. (b) Immunofluorescence than a non–ST-17 strain (Fig. 5 a), HvgA strongly contributes analysis was performed with rabbit polyclonal anti-HvgA antibodies (pro- to this ST-17–specic fi phenotype (Fig. 5 b), and the non–ST-17 tein fragment 30–216) revealed with an anti-IgG coupled to Alexa Fluor allelic variant BibA has no signic fi ant impact on gut col onization 488. Bars, 10 µm. (c) Flow cytometry analysis of L. lactis expressing HvgA (Fig. 5 c). Ex vivo experiments with cecal tissue explants also incubated with a polyclonal anti-HvgA antibody and stained with an demonstrated an HvgA-mediated bacterial association to the Alexa Fluor 488–conjugated anti–rabbit IgG antibody (black line histogram). (d) Comparison of adhesive properties of L. lactis and HvgA- or BibA- cecal epithelium (Fig. 5 d). In addition, competition experi- expressing strains on Caco-2 and hCMEC/D3 cell lines. Error bars repre- ments for intestinal colonization in which 5 × 10 CFUs of sent the SD. *, Mann-Whitney test (P < 0.05), relative to L. lactis + vector. each strain were simultaneously inoculated indicated that within (e) hvgA confers to WT non–ST-17 and ST-17 adhesion phenotype. Com- <1 wk, ST-17 WT totally out-competes a non–ST-17 WT parison of adhesive properties of WT non–ST-17 and bibA mutant or strain (Fig. 5 e), as well as an isogenic hvgA mutant (Fig. 5 f). complemented strains expressing BibA or HvgA on brain microvascular Together, these results demonstrate that HvgA confers a selec- endothelial cell line hCMEC/D3. Experiments were performed in triplicate tive advantage at this initial step of infection. and repeated independently at least three times; graphs show cumulative We next addressed whether HvgA could provide the data from all experiments. **, Mann-Whitney test (P < 0.01), relative to WT ST-17 strain with an enhanced ability to translocate across the non ST-17. 2318 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t Figure 5. HvgA enables GBS persistent intestinal colonization and promotes its crossing of the intestinal barrier. (a–c) Groups (n = 6) of 3–4-wk-old Swiss female mice were infected orally with 10 CFUs WT GBS or mutant strains, and fecal shedding was assessed 3, 7, and 14 d after inoculation by CFUs enumeration. Values represent fecal shedding of the six mice in a cage. (d) Infec- tion of Swiss mice (n = 4) ligated cecum with 10 GBS WT ST-17 or hvgA mutant strain. Bacteria were recovered 1 h after infection. Values are expressed as the percentage of adhesion relative to the inoculum. (e and f) Competition assays between WT ST-17 (BM110) and WT non–ST-17 (NEM316) or hvgA BM110 mutant. 3–4-wk-old Swiss mice (n = 6) were infected orally with a 1:1 mixture of the two strains (total dose 10 CFUs). Bacteria were enumerated from the feces collected 3, 7, and 10 d after infection. (g) Mortality rate 12 h after infection of 15–21 d old BALB/c mice (n = 10) infected orally with the WT ST-17 or hvgA mutant strains. (h) Groups of 4-wk-old BALB/c mice (n = 10) were inoculated orally with 10 CFUs WT ST-17 or hvgA mutant strains. 8 h after infection, animals were sacrificed. Cecum were collected and divided longitudinally in two parts. One half was directly homogenized: extra- and intratissular bacteria associated with the cecum were enumerated (no genta.). The second half was incubated for 3 h in DMEM containing 250 µg/ml gentamicin to kill extratissular bacteria and homogenized. Intratissular invading bacteria were then enumerated (genta.). Animal experiments represented in this figure were repeated at least two times and groups of mice contained at least five animals. Error bars represent the SD of depicted variable performed in triplicate. Asterisks indicate significant differences as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). after oral inoculation of 4–5-wk-old BALB/c mice with 10 microscopy (Fig. S2). In mice with the highest WT-ST17 bac- 9 7 or 10 , no mortality was observed. But here again, the WT terial load (5 × 10 CFUs) in the brain, GBS were observed ST-17 strain adhered to and invaded the cecal tissue more adhering to the inner meningeal envelopes (pia matter, Fig. S2 a) significantly than the hvgA mutant (Fig. 5 h and Fig. S1 a), in the brain microvessel (Fig. S2 b), in the brain parenchyma indicating a key role for HvgA in GBS ST-17 ability to cross (Fig. S2 c), or in the choroid plexuses (Fig. S2 d). In contrast, the intestinal barrier. Moreover, in germ-free animals infected no bacterium was observed in mice infected with the hvgA with WT GBS ST-17, bacteria could be observed adhering mutant. To focus specifically on the crossing of the BBB and to the enterocytes and in the lamina propria (Fig. S1, b-e), bypass the HvgA phenotype at the BALB/c intestinal barrier similar to what was observed in the human previously de- level, we developed a CNS infection model in which pre- scribed LOD case (Fig. 1, a and b) weaning 3 wk mice were infected i.v. every 12 h with a rela- tively low inoculum (5 × 10 CFUs) to avoid unspecific BBB HvgA contributes to GBS crossing of the blood–brain barrier opening caused by the massive systemic inflammation trig - and the onset of meningitis gered by high bacterial loads, but to instead mimic blood- We next analyzed HvgA contribution to central nervous system borne neonatal meningitis. Monitoring of bacterial loads in (CNS) invasion. A role for HvgA in this process was supported the blood and the CNS showed that whereas WT ST-17 and by results obtained with orally inoculated animals: 15–21-d-old its hvgA isogenic mutant induce a similar level of bacteremia BALB/c mice infected with the WT GBS-ST17 strain have 48 h after i.v. inoculation (Fig. 6 c), hvgA is significantly im - significantly higher bacterial counts in the brain than the paired in its ability to invade the CNS (Fig. 6 d). In agreement mice infected with the hvgA mutant at the experiment end- with these quantitative data, real-time imaging of biolumines- point (Fig. 6, a and b). Moreover, animals that died in the cent bacteria along the infectious process revealed a marked meantime are those for which bacterial counts in the brain bioluminescence emission in the head of animals infected are the highest (Fig. 6 a), arguing for a causal relationship with WT ST-17 (4/4 animals), which was detectable 20 h after between HvgA expression and GBS ST-17 ability to reach infection and is visible until the death of the animal, whereas the CNS after oral inoculation. To investigate the onset of merely no signal was detected in mice infected with the meningitis in these orally inoculated animals, their brains hvgA mutant (Fig. 6 e). Neuropathological analysis of in- were examined by immunohistochemistry and confocal fected animals 48 h after i.v. inoculation disclosed an intense JEM VOL. 207, October 25, 2010 2319 Figure 6. HvgA enables GBS crossing of the BBBs. (a and b) Groups of 15–21 d old BALB/c mice (n > 8) 9 8 were inoculated orally with 10 (a) or 5 × 10 CFUs (b) WT ST-17 or hvgA mutant strain and brain invasion was assessed 12-h after inoculation by CFUs enumeration. (c and d) Groups of 3–4-wk-old BALB/c mice (n > 8) were infected by repeated i.v. injections of 5 × 10 CFUs every 12 h and sacrificed at 48 h, and bacteria enumerated in blood (c) and brain (d). Data shown are the results of three independent experiments. (e) Real-time imaging of CNS infection. Groups of BALB/c mice (n = 4) were in- fected i.v. with 10 CFUs every 12 h. Bioluminescence was assessed every 12 h (the images shown correspond to the 36 h after injection time point). (f–h) Histopathologi- cal examination (Gram staining) of CNS tissue samples from WT-ST-17 infected mice reveals Gram-positive cocci in the meninges (f), in the choroid plexuses (g), in brain microvessel endothelial cells and the surrounding brain parenchyma (h). (i–k) Immunofluorescence staining of brain sections obtained from animals infected with the WT ST-17 in d, showing infection of meninges (i), sub- meningeal space (j), and brain microvessel endothelial cells (k). Bars: (f–h) 25 µm; (i–5) 20 µm. All experiments were repeated at least three times. NS, not significant. Asterisks indicate significant differences as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). associated with ST-17 GBS isolates: (1) their close association with LOD, which can now be linked to HvgA-mediated intestinal colonization and subsequent crossing of the intestinal barrier, and (2) their close association with meningitis, which can now be linked to the HvgA-mediated cross- ing of the BBB. This study den fi itely establishes the hypervirulence of the ST-17 GBS clone and links it to enhanced intestinal colonization and barrier breaching potentials. Importantly, it illustrates how a microbial factor implicated in bacterial intestinal colonization can behave as a specific virulence factor in the neonatal con - text, at a time when the intestinal microflora is not yet established and cannot exerts its buffer - WT ST-17 streptococcal infection of choroid plexuses and ing effect on potential pathogenic bacteria such as GBS. Given meninges (Fig. 6, f, g, i, and k), as well as in brain microvessels the burden of ST-17–associated LOD and neonatal meningi- and the surrounding brain parenchyma (Fig. 6, h and k), a result tis (Poyart et al., 2008; CDC, 2009), hvgA and its gene product are matching our observations in the human LOD case associated now promising targets for developing diagnostic tools (Lamy with ST-17 GBS (Fig. 1, c–l). Together, these in vivo results dem- et al., 2006) and vaccines (Santi et al., 2009), respectively. onstrate HvgA contribution in ST-17 access to the CNS, a result that corroborates our in vitro data (Fig. 3, c and d). MATERIAL AND METHODS Bacterial strains, genetic constructions, and growth conditions. The Conclusions main characteristics of bacterial strains and plasmids used in this study are listed in Table S1. GBS NEM316, capsular serotype III, and MLST type ST Using complementary approaches, we have uncovered a key 23, and GBS BM110, capsular serotype III, and MLST sequence type ST-17 determinant of the pathophysiology of ST-17–associated are well-characterized isolates from human with invasive infections. GBS LOD. We have identified an ST-17–specific surface-expressed clinical strains from invasive infection were collected by the National Refer- protein, HvgA, and demonstrated that it is a major determi- ence Center for Streptococci from 2006 and 2009. GBS mutants were nant of ST-17 hypervirulence in neonates. Our results pro- constructed by in-frame deletions in bibA (GBS NEM316), hvgA (GBS vide a rational explanation for the two main characteristics BM110), and srtA (GBS BM110), as previously described (Dramsi et al., 2006) 2320 Hypervirulence of ST-17 group B streptococcus | Tazi et al. B r i e f D e f i n i t ive R e p o r t using primers listed in Table S3 and Fig. 2 a. The expected in-frame dele- injected intragastrically. At the indicated times, cecum were collected, rinsed tions were confirmed by PCR and sequence analysis. Complemented GBS in DME, and divided in two parts longitudinally. One half was directly ho- mutant strains and L. lactis expressing BibA or HvgA were constructed as mogenized in PBS with a tissue homogenizer. The second half was incu- follows. bibA and hvgA genes were amplified by PCR from chromosomal bated for 3 h in DME containing 250 µg/ml gentamicin to determine DNA of GBS NEM316 and BM110, respectively, using primers O5 and O6 bacterial invasion, rinsed, and homogenized. Serial dilutions were plated on (Fig. 2 a and Table S3) and cloned into the pOri23 shuttle vector. Recombi- selective medium (Granada) for CFU counts. For cecum-ligated loop ex- nant plasmids PbibA and PhvgA were introduced by electroporation into periments, animals were anesthetized. The whole cecums were closed at GBS mutants and L. lactis. Unless otherwise specified, GBS and L. lactis were both ends with surgical sutures. 200 µl of the adequate bacterial dilution was cultured at 37°C in Todd Hewitt (TH) broth or agar and antibiotics were injected in the lumen. 1 h after infection, the mice were sacric fi ed; the cecum- used at the following concentrations: erythromycin, 10 µg.ml ; kanamycin, ligated loops were excised and opened longitudinally. For competitive assay, 1,000 µg.ml . both WT and isogenic mutants were mixed in a 1:1 ratio oral infection ex- periments. At the indicated times, stools were collected and serial dilutions Generation of anti-BibA and anti-HvgA rabbit polyclonal anti- were plated on selective medium. i.v. infections were performed by i.v. in- bodies. Recombinant Gbs2018 segments were expressed and purie fi d as fol - jection every 12 h for 36 h. All of the procedures were in agreement with lows. DNA fragments intragenic to bibA (nt 100–1185) and hvgA (nt 88–648) the guidelines of the European Commission for the handling of laboratory were produced by PCR using genomic DNA of GBS NEM316 and BM110, animals, directive 86/609/EEC (http://ec.europa.eu/environment/chemicals/ respectively, as templates, as well as the primers listed in Table S3. DNA frag- lab_animals/home_en.htm) and were approved by the Animal Care and Use ments were digested with the appropriate enzymes and cloned into pET- Committees of the Institut Pasteur. 26b(+) and pET2817, respectively. The resulting plasmids were introduced into E. coli BL21DE3/pDIA17 for protein expression. Recombinant proteins Bioluminescence real-time imaging. GBS WT-ST17 strain and the were purie fi d under native conditions on Ni-NTA columns (QIAGEN), fol - hvgA isogenic mutant were made bioluminescent after introduction plas- lowed by Q–Sepharose anion exchange chromatography (GE Healthcare). mid pTCVlux by electroporation (Table S1). After i.v. infection, BALB/c The purie fi d BibA or HvgA truncated proteins were injected into rabbit to mice bioluminescence was monitored each day, as previously described produce antibodies, as previously described (Lalioui et al., 2005). (Disson et al., 2009). 12 h before infection and every 12 h during the infec- tion, animals were treated with erythromycin (20 mg/kg ). RNA isolation, reverse transcription, and qRT-PCR. Total RNAs were extracted from bacteria as previously described (Lamy et al., 2004). For Tissue labeling. Thin sections, thick sections, and whole-mount tissue la- isolation of bacterial RNA from human blood, bacteria were grown in TH beling were performed as previously described (Disson et al., 2009). Acquisi- broth to the mid-logarithmic phase (OD 0.3–0.4), washed in PBS, and tion of images was realized with an upright confocal microscope (Carl Zeiss, 600nm resuspended in an equal volume of freshly drawn human blood from non- Inc.), using a 40× water immersion objective for the whole-mount tissue or immune healthy donors and incubated for 1 h at 37°C. RNA isolation from an oil immersion objective for the thick section labeling. Reconstructions animal blood and tissues was as previously described (Oggioni et al., 2006). were realized using Imaris software. cDNA synthesis was performed on DNase-I–treated RNA (50 ng of two Online supplemental material. Fig. S1 shows the role of HvgA in the independent extracts) with specific reverse primers using SuperScript II re - crossing of the intestinal barrier. Fig. S2 shows that GBS ST-17 crosses the verse transcription (Invitrogen). qRT-PCR analysis was performed as previ- BBB upon oral inoculation. Table S1 describes the bacterial strains and plas- ously described (Yamamoto et al., 2005) using primers listed in Table S3. 2Ct mids used in this study. Table S2 describes the origin, the serotype, and the The relative fold change of each gene was calculated from 2 by using ST of GBS strains used in this study. Table S3 shows the list of primers used rpoB as an internal control gene. for genetic constructions and qRT-PCR. Online supplemental material is Immunoblots and bacterial immunofluorescence stainings. For anal- available at http://www.jem.org/cgi/content/full/jem.20092594/DC1. ysis of HvgA expression, cell wall proteins were extracted as previously de- scribed (Dramsi et al., 2006, Lalioui et al., 2005). After SDS-PAGE, proteins We are grateful to Alexandra Gruss for helpful discussion and critical reading of the were transferred to nitrocellulose membrane. HvgA was detected using manuscript. We thank Pierre-Olivier Couraud for providing HCMEC/D3 cells. This work was supported by the Agence Nationale de la Recherche (ANR rabbit-specific pAb and horseradish peroxidase–coupled anti–rabbit second - HyperVirGBS Project; ANR-08-MIE-015), Institut National de la Santé et de la ary antibodies and the ECL Reagent (GE Healthcare). Immunofluorescence Recherche Médicale, Institut Pasteur, Fondation pour la Recherche Médicale (FRM), staining and fluorescence-activated cell sorter analysis were carried out as Université Paris Descartes, the Institut de Veille Sanitaire, Programme Transversal de previously described (Dramsi et al., 2006). Recherche #190. S. Bellais was a recipient of a post-doctoral fellowship from the ANR-08-MIE-015 grant and O. Disson from FRM and Institut National de la Santé Cell cultures assays. Human epithelial (Caco-2, A549, and HeLa), and et de la Recherche Médicale. human endothelial (HUVEC) cell lines are from the American Type Culture The authors declare that they have no competing financial interest. Collection. Human brain endothelial cell line hCMEC/D3 (Weksler et al., 2005) was provided by P.O. Couraud (Institut National de la Santé et de la Submitted: 4 December 2009 Recherche Médicale, 75014 Paris, France). 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The Journal of Experimental Medicine – Pubmed Central

Published: Oct 25, 2010

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