Search

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase., (© 2023 Federation of European Biochemical Societies.)

Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: In infants, the mode of acquisition of CC17 group B Streptococcus (GBS), the hypervirulent clone responsible for late-onset disease (LOD), remains elusive., Methods: In a prospective multicenter study in France, we evaluated GBS colonization in mother-baby pairs with 2 months of follow-up between 2012 and 2015. Criteria included positivity for GBS colonization at antenatal screening or at delivery. Maternal vaginal samples and infant oral cavity and stool samples were analyzed at delivery, 21 ± 7 days (D21), and 60 ± 7 days (D60) post-delivery., Results: A total of 890 mother-baby pairs were analyzed. GBS colonized 7%, 21%, and 23% of the infants at birth, D21, and D60, respectively, of which 10%, 11%, and 13% were identified as CC17 GBS. Concordance between maternal and infant GBS type was 96%. At D21, the main risk factors for infant colonization by GBS were simultaneous maternal colonization of the vagina (odds ratio [OR], 4.50; 95% confidence interval [CI], 1.69-15.61) and breast milk (OR, 7.93; 95% CI, 3.81-17.14). Importantly, 38% (95% CI, 23%-56%) of infants colonized by CC17 GBS appeared colonized for the first time at D60 vs 18% (95% CI, 14%-24%; P < .049) of infants colonized by non-CC17 GBS. Multivariate analysis showed a higher risk for de novo infant colonization by CC17 at D60 than by other GBS (OR, 2.45; 95% CI, 1.02-5.88)., Conclusions: The high incidence of CC17 GBS in LOD is likely due to an enhanced post-delivery mother-to-infant transmission., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The core PI-2b pilus present in "hypervirulent" ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin α v subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS., (© 2018 John Wiley & Sons Ltd.)

Streptococcus agalactiae (Group B Streptococcus or GBS) is a frequent cause of serious disease in newborns and adults. Epidemiological evidence indicates a strong association between GBS strains belonging to the hypervirulent CC17 clonal complex and the occurrence of meningitis in neonates. We investigate here the role of PbsP, a cell wall plasminogen binding protein, in colonization of the central nervous system by CC17 GBS. Deletion of pbsP selectively impaired the ability of the CC17 strain BM110 to colonize the mouse brain after intravenous challenge, despite its unchanged capacity to persist at high levels in the blood and to invade the kidneys. Moreover, immunization with a recombinant form of PbsP considerably reduced brain infection and lethality. In vitro, pbsP deletion markedly decreased plasmin-dependent transmigration of BM110 through brain microvascular endothelial cells. Although PbsP was modestly expressed in bacteria grown under standard laboratory conditions, pbsP expression was markedly upregulated during in vivo infection or upon contact with cultured brain endothelial cells. Collectively, our studies indicate that PbsP is a highly conserved Plg binding adhesin, which is functionally important for invasion of the central nervous system by the hypervirulent CC17 GBS. Moreover, this antigen is a promising candidate for inclusion in a universal GBS vaccine.

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5' promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110ΔcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential., Competing Interests: The authors have declared that no competing interests exist.

Group B Streptococcus (GBS) is the leading cause of neonatal invasive infections and an emerging pathogen in the elderly. Our objectives were to describe the evolution of GBS resistance to antibiotics in France and to investigate the emergence of fluoroquinolone (FQ)-resistant isolates. A total of 8,757 unrelated GBS isolates were collected and tested for antibiotic susceptibility from 2007 to 2014 according to EUCAST recommendations. All isolates were susceptible to penicillin G, amoxicillin, and vancomycin. Resistance to macrolides decreased from 47.0% to 30.0%, whereas high-level resistance to aminoglycosides, especially amikacin, increased from 6.4% to 8.8% and 24 isolates (0.3%) were highly resistant to gentamicin. FQ resistance gradually increased from 0.2% in 2007 (n = 1) to 1.5% in 2014 (n = 18, P < 0.01). Capsular polysaccharide (CPS) genotyping, multilocus sequence typing, and sequencing of the quinolone resistance-determining region (QRDR) showed that GBS isolates of sequence type 19 (ST-19) CPS type V were largely overrepresented in FQ-resistant isolates (n = 30, 45.5%). All 30 strains displayed the same QRDR mutations and were often associated with cross-resistance to macrolides (93.3%) and gentamicin (30%). In conclusion, we report the rise of FQ- and aminoglycoside-resistant GBS in France over an 8-year study period, an evolution likely linked to the clonal expansion of ST-19 CPS V-resistant isolates. This study emphasizes the need for a continuous surveillance of GBS epidemiology and antibiotic susceptibility., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Induction of type I interferon (IFN) in response to microbial pathogens depends on a conserved cGAS-STING signaling pathway. The presence of DNA in the cytoplasm activates cGAS, while STING is activated by cyclic dinucleotides (cdNs) produced by cGAS or from bacterial origins. Here, we show that Group B Streptococcus (GBS) induces IFN-β production almost exclusively through cGAS-STING-dependent recognition of bacterial DNA. However, we find that GBS expresses an ectonucleotidase, CdnP, which hydrolyzes extracellular bacterial cyclic-di-AMP. Inactivation of CdnP leads to c-di-AMP accumulation outside the bacteria and increased IFN-β production. Higher IFN-β levels in vivo increase GBS killing by the host. The IFN-β overproduction observed in the absence of CdnP is due to the cumulative effect of DNA sensing by cGAS and STING-dependent sensing of c-di-AMP. These findings describe the importance of a bacterial c-di-AMP ectonucleotidase and suggest a direct bacterial mechanism that dampens activation of the cGAS-STING axis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium., (© 2016 John Wiley & Sons Ltd.)

Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to β-hemolytic streptococci., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Group B Streptococcus (GBS), a commensal organism, can turn into a life-threatening pathogen in neonates and elderly, or in adults with severe underlying diseases such as diabetes. We developed a vaccine targeting the GBS glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme detected at the bacterial surface, which was proven to be effective in a neonatal mouse model of infection. Since this bacterium has emerged as an important pathogen in non-pregnant adults, here we investigated whether this vaccine also confers protection in an adult susceptible and in a diabetic mouse model of infection. For immunoprotection studies, sham or immunized adult mice were infected with GBS serotype Ia and V strains, the two most prevalent serotypes isolated in adults. Sham and vaccinated mice were also rendered diabetic and infected with a serotype V GBS strain. For toxicological (pre-clinical) studies, adult mice were vaccinated three times, with three concentrations of recombinant GAPDH adjuvanted with Allydrogel, and the toxicity parameters were evaluated twenty-four hours after the last immunization. For the stability tests, the vaccine formulations were maintained at 4°C for 6 and 12 months prior immunization. The results showed that all tested doses of the vaccine, including the stability study formulations, were immunogenic and that the vaccine was innocuous. The organs (brain, blood, heart, and liver) of vaccinated susceptible or diabetic adult mice were significantly less colonized compared to those of control mice. Altogether, these results demonstrate that the GAPDH-based vaccine is safe and stable and protects susceptible and diabetic adult mice against GBS infections. It is therefore a promising candidate as a global vaccine to prevent GBS-induced neonatal and adult diseases.

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

The Group B Streptococcus (GBS) 'hypervirulent' ST-17 clone is strongly associated with invasive neonatal meningitis. Comparative genome analyses revealed that the serine-rich repeat (Srr) glycoprotein Srr2 is a cell wall-anchored protein specific for ST-17 strains, the non-ST-17 isolates expressing Srr1. Here, we unravel the binding capacity of GBS Srr proteins to relevant components of the host fibrinolysis pathway. We demonstrate that: (i) Srr2 binds plasminogen and plasmin whereas Srr1 does not; (ii) the ability of ST-17 strains to bind fibrinogen reflects a high level surface display of Srr2 combined with a higher affinity of Srr2 than Srr1 to bind this ligand; and (iii) Srr2 binding to host plasma proteins results in the formation of bacterial aggregates that are efficiently endocytosed by phagocytes. Importantly, we show that Srr2 increased bacterial survival to phagocytic killing and bacterial persistence in a murine model of meningitis. We conclude that Srr2 is a multifaceted adhesin used by the ST-17 clone to hijack ligands of the host coagulation system, thereby contributing to bacterial dissemination and invasiveness, and ultimately to meningitis., (© 2015 John Wiley & Sons Ltd.)

Background: Streptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing., Results: Our study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively., Conclusions: This comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.

Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.

Group B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the "pellicle") overlaid by a 15-45 nm thick layer of CPS (the "capsule"). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Among 1,827 group B Streptococcus (GBS) strains collected between 2006 and 2013 by the French National Reference Center for Streptococci, 490 (26.8%) strains were erythromycin resistant. The erm(T) resistance gene was found in six strains belonging to capsular polysaccharides Ia, III, and V and was carried by the same mobilizable plasmid, which could be efficiently transferred by mobilization to GBS and Enterococcus faecalis recipients, thus promoting a broad dissemination of erm(T)., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Streptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine.

The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

The two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates., Biological Significance: We believe that this manuscript will be of interest to Journal of Proteomics readers since the paper describes the identification of several putative virulence factors and vaccine candidates of the group B streptococcus, an important pathogen, using a simple proteomics strategy involving LC-MS analysis of culture supernatants obtained from two strains with divergent gene expression patterns. This technique provided the most comprehensive inventory of extracellular proteins obtained from a single streptococcal species thus far. The approach described has the added benefit of being easily applicable to a large number of different strains, making it ideal for the identification of conserved vaccine candidates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.

Group B Streptococcus (GBS) is the leading cause of meningitis in neonates. We have previously shown that plasminogen, once recruited to the GBS cell surface and converted into plasmin by host-derived activators, leads to an enhancement of bacterial virulence. Here, we investigated whether plasmin(ogen) bound at the GBS surface contributes to blood-brain barrier penetration and invasion of the central nervous system. For that purpose, GBS strain NEM316 preincubated with or without plasminogen plus tissue type plasminogen activator was analyzed for the capacity to adhere to, invade and transmigrate the human brain microvascular endothelial cell (hBMEC) monolayer, and to penetrate the central nervous system using a neonatal mouse model. At earlier times of infection, plasmin(ogen)-treated GBS exhibited a significant increase in adherence to and invasion of hBMECs. Later, injury of hBMECs were observed with plasmin(ogen)-treated GBS that displayed a plasmin-like activity. The same results were obtained when hBMECs were incubated with whole human plasma and infected with untreated GBS. To confirm that the observed effects were due to the recruitment and activation of plasminogen on GBS surface, the bacteria were first incubated with epsilon-aminocaproic acid (εACA), an inhibitor of plasminogen binding, and thereafter with plasmin(ogen). A significant decrease in the hBMECs injury that was correlated with a decrease of the GBS surface proteolytic activity was observed. Furthermore, plasmin(ogen)-treated GBS infected more efficiently the brain of neonatal mice than the untreated bacteria, indicating that plasmin(ogen) bound to GBS surface may facilitate the traversal of the blood-brain barrier. A higher survival rate was observed in offspring born from εACA-treated mothers, compared to untreated mice, and no brain infection was detected in these neonates. Our findings suggest that capture of the host plasmin(ogen) by the GBS surface promotes the crossing of the blood-brain barrier and contributes to the establishment of meningitis.

Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS.

Background: The capsular polysaccharide (CPS) is an important virulence factor and a vaccine target of the major neonatal pathogen group B Streptococcus (GBS). Population studies revealed no strong correlation between CPS type and multilocus sequence typing (MLST) cluster, with the remarkable exception of the worldwide spread of hypervirulent GBS CC17, which were all until recently CPS type III., Methods: A total of 965 GBS strains from invasive infection isolated in France were CPS typed and the presence of the CC17-specific surface protein encoding gene hvgA gene was investigated. Three hvgA-positive GBS strains screened were surprisingly CPS type IV and thus further characterized by MLST typing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing., Results: MLST and PFGE demonstrated a capsular switching from CPS type III to IV within the highly homogeneous GBS CC17. Sequence analysis revealed that this capsular switch was due to the exchange of a 35.5-kb DNA fragment containing the entire cps operon., Conclusions: This work shows that GBS CC17 hypervirulent strains have switched one of their main vaccine targets. Thus, continued surveillance of GBS population remains of the utmost importance during clinical trials of conjugate GBS vaccines.

Streptococcus agalactiae (Group B Streptococcus, GBS) is the major cause of severe bacterial disease and meningitis in newborns. The zebrafish (Danio rerio) has recently emerged as a valuable and powerful vertebrate model for the study of human streptococcal infections. In the present study we demonstrate that adult zebrafish are susceptible to GBS infection through the intraperitoneal and intramuscular routes of infection. Following intraperitoneal challenge with GBS, zebrafish developed a fulminant infection 24-48 h post-injection, with signs of pathogenesis including severe inflammation at the injection site and meningoencephalitis. Quantification of blood and brain bacterial load confirmed that GBS is capable of replicating in the zebrafish bloodstream and penetrating the blood-brain barrier, resulting in the induction of host inflammatory immune responses in the brain. Additionally, we show that GBS mutants previously described as avirulent in the mice model, have an impaired ability to cause meningitis in this new in vivo model. Taken together, our data demonstrates that adult zebrafish may be used as a bacterial meningitis model as a means for deciphering the pathogenesis and development of invasive GBS disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Group B Streptococcus (GBS) is the leading cause of neonatal septicemia and meningitis. Pili appendages were shown to play a critical role in bacterial adhesion and colonization of human tissues. Recently it was claimed that binding of the pilus-associated adhesin PilA to collagen is a critical, initial step in promoting interactions with the α2β1 integrin expressed on brain endothelial cells. Here we show that strain NCTC10/84 used in this study is not representative for GBS isolates and question the importance of collagen as a critical extracellular matrix component for GBS infections of the central nervous system., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Rapid adaptation to changing environments is key in determining the outcome of infections caused by the opportunistic human pathogen Streptococcus agalactiae. We previously demonstrated that the RofA-like protein (RALP) regulators RogB and Rga activate their downstream divergently transcribed genes, that is, the pilus operon PI-2a and the serine-rich repeat encoding gene srr1, respectively. Characterization of the Rga regulon by microarray revealed that the PI-2a pilus was strongly controlled by Rga, a result confirmed at the protein level. Complementation experiments showed that the expression of Rga, but not RogB, in the double ΔrogB/Δrga mutant, or in the clinical strain 2603V/R displaying frameshift mutations in rogB and rga genes, is sufficient to restore wild-type expression levels of PI-2a pilus and Srr1. Biofilm formation was impaired in the Δrga and Δrga/rogB mutants and restored on complementation with rga. Paradoxically, adherence to intestinal epithelial cells was unchanged in the Δrga mutant. Finally, the existence of several clinical isolates mutated in rga highlights the concept of strain-specific regulatory networks.

Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli.

Group B Streptococcus (GBS) is a frequent agent of life-threatening sepsis and meningitis in neonates and adults with predisposing conditions. We tested the hypothesis that activation of the inflammasome, an inflammatory signaling complex, is involved in host defenses against this pathogen. We show in this study that murine bone marrow-derived conventional dendritic cells responded to GBS by secreting IL-1β and IL-18. IL-1β release required both pro-IL-1β transcription and caspase-1-dependent proteolytic cleavage of intracellular pro-IL-1β. Dendritic cells lacking the TLR adaptor MyD88, but not those lacking TLR2, were unable to produce pro-IL-1β mRNA in response to GBS. Pro-IL-1β cleavage and secretion of the mature IL-1β form depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) sensor and the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain adaptor. Moreover, activation of the NLRP3 inflammasome required GBS expression of β-hemolysin, an important virulence factor. We further found that mice lacking NLRP3, apoptosis-associated speck-like protein, or caspase-1 were considerably more susceptible to infection than wild-type mice. Our data link the production of a major virulence factor by GBS with the activation of a highly effective anti-GBS response triggered by the NLRP3 inflammasome.

Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC) is a peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.

Streptococcus agalactiae (or group B streptococcus; GBS) is a leading cause of neonatal morbidity and mortality in the developed countries. Several epidemiological typing tools have been developed for GBS to investigate the association between genotype and disease and to assess genetic variations within genogroups. This study compared the semi-automated repetitive sequence-based PCR Diversilab® system (DL) with MLST and pulsed field gel electrophoresis (PFGE) for determining the relatedness of invasive GBS strains. We analysed 179 unrelated GBS strains isolated from adult (n=108) and neonatal (n=71) invasive infections. By MLST, strains were resolved into 6 clonal complexes (CCs) including 23 sequence-types (STs), and 4 unique STs, whereas DL differentiated these isolates into 12 rep-PCR clusters (rPCs) and 9 unique rep-PCR types. The discriminatory power of both methods was similar, with Simpson's diversity indexes of 71.9% and 70.6%, respectively. However, their clustering concordance was low with Wallace concordance coefficients inferior to 0.4. PFGE was performed on 31 isolates representative of the most relevant DLrPCs clustered within the 3 major MLST CCs (CC-17, CC-23 and CC-1). As already observed with MLST, the concordance of DL with PFGE was low for all three CCs (Wallace coefficient <0.5), PFGE being more discriminative than rep-PCR. In summary, this work suggests that DL is less appropriate than MLST or PFGE to study GBS population genetic diversity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone., Methodology/principal Findings: In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity., Conclusions/significance: These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.

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 histopathological 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.

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a K(d) of 0.5 microm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.

In streptococci, the secA2 locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The secA2 locus of Streptococcus agalactiae strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.

We describe in Streptococcus agalactiae an atypical family of conjugative transposons named TnGBSs which associates DDE transposition and conjugation. We present evidence that the transposition of TnGBS2, the prototype of this family, is catalysed by a new class of DDE transposases that are widespread in Gram-positive bacteria. Remarkably, transposition occurs in intergenic regions, 15 or 16 bp upstream the -35 sequence of promoters, minimizing the burden on the host cell and suggesting an association between transcription and transposition. Transposition catalyses the formation of a circular intermediate that is substrate for subsequent conjugative intercellular transfer. Conjugation is initiated at an origin of transfer by a transposon-encoded relaxase. Whereas all integrative and conjugative elements described so far encode a phage-related integrase, TnGBS2 is the first example of conjugative transposon whose recombination is mediated by a DDE transposase. The combination of DDE transposition with conjugation implies recombination constraints linked to the physical separation of donor and recipient molecules.

Type I interferon (IFN) is an important host defense cytokine against intracellular pathogens, mainly viruses. In assessing IFN production in response to group B streptococcus (GBS), we find that IFN-beta was produced by macrophages upon stimulation with both heat-killed and live GBS. Exposure of macrophages to heat-killed GBS activated a Toll-like receptor (TLR)-dependent pathway, whereas live GBS activated a TLR/NOD/RIG-like receptor (RLR)-independent pathway. This latter pathway required bacterial phagocytosis, proteolytic bacterial degradation, and phagolysosomal membrane destruction by GBS pore-forming toxins, leading to the release of bacterial DNA into the cytosol. GBS DNA in the cytosol induced IFN-beta production via a pathway dependent on the activation of the serine-threonine kinase TBK1 and phosphorylation of the transcription factor IRF3. Thus, activation of IFN-alpha/-beta production during infection with GBS, commonly considered an extracellular pathogen, appears to result from the interaction of GBS DNA with a putative intracellular DNA sensor or receptor.

Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.

Clinical features and molecular characterization of 109 group B streptococci causing neonatal invasive infections were determined over an 18-month period in France. Sixty-four percent of the strains were from late-onset infections, and 75% were capsular type III. The hypervirulent clone ST-17 was recovered in 80% of meningitis cases.

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.