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Abstract: 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 &y& Elsevier]

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer. S. gallolyticus is part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis. S. gallolyticus has the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore, S. gallolyticus synthesizes all 20 amino acids and more vitamins than any other sequenced Streptococcus species. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antimicrobial drugs targeting the reportedly essential type II fatty acid synthesis (FASII) pathway have been recently acclaimed for their efficacy against infections caused by multiresistant Gram-positive bacteria. Our findings show that the strategy for antibiotic development based on FASII pathway targets is fundamentally flawed by the fact that exogenous fatty acids fully bypass inhibition of this pathway in both in vitro and in vivo conditions. We demonstrate that major Gram-positive pathogens—such as streptococci, pneumococci, enterococci and staphylococci—overcome drug-induced FASII pathway inhibition when supplied with exogenous fatty acids, and human serum proves to be a highly effective source of fatty acids. For opportunist pathogen Streptococcus agalactiae, growth in serum leads to an overall decrease of FASII gene expression. No antibiotic inhibitor could have a stronger effect than the inactivation of the target gene, so we challenged the role of FASII using deletion mutants. Our results unequivocally show that the FASII target enzymes are dispensable in vivo during S. agalactiae infection. The results of this study largely compromise the use of FASII-based antimicrobials for treating sepsis caused by Gram-positive pathogens. [ABSTRACT FROM AUTHOR]

We describe in Streptococcus agalactiae an atypical family of conjugative transposons named Tn GBSs which associates DDE transposition and conjugation. We present evidence that the transposition of Tn GBS2, 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, Tn GBS2 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. [ABSTRACT FROM AUTHOR]

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 evolution- ary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosornal 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. [ABSTRACT FROM AUTHOR]

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae[group B streptococcus (GBS)] is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1-C2 and srtC3-C4, with a similar genetic organization and are thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG protein encoding genes, two upstream and one downstream, and a divergently transcribed regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3-C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3-C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus-associated adhesin. Surprisingly, pilus-like structures can be formed in the absence of the two minor components, i.e. the putative anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Gram-positive pathogens. [ABSTRACT FROM AUTHOR]

In this study, we carried out a detailed structural and functional analysis of aStreptococcus agalactiae(GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system ofStreptococcus pyogenes. In GBS,covRandcovSare part of a seven gene operon transcribed from two promoters that are not regulated by CovR. AΔcovSRmutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of theΔcovSRmutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, theΔcovSRmutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD50 of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of theΔcovSRmutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses. [ABSTRACT FROM AUTHOR]

Summary D-alanylation of lipoteichoic acid (LTA), allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D-alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA[sup –] isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal-derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow-derived macrophages and human neutrophils than the wild-type strain. In addition, the virulence of the DltA[sup –] mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild-type strain from the lung of three-week-old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three-week-old mice, the survival of the DltA[sup –] mutant is markedly reduced in the blood in comparison to that of the wild-type strain. We hypothesize that the decreased virulence of the DltA[sup –] mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D-alanylation of LTA contributes to the virulence of S. agalactiae . [ABSTRACT FROM AUTHOR]

The main causes of microbial death after heat exposure are not well understood. Here, it is shown that the heat-shock protein CIpP plays a major role in heat-induced growth arrest in Streptococcus agalactiae. A mutant lacking the CIpP protease was more sensitive to the inhibitory effects of heat, salt and oxidative stress than the isogenic wild-type strain. During growth arrest, this mutant displayed important modifications of its total protein content, including a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Analysis of protein carbonylation demonstrated that the CIpP protease plays a role in preventing accelerated protein oxidation. Higher levels of oxidized DnaK, a key modulator of the heat-shock regulon, were observed in the CIpP mutant and these were increased following heat shock. Accumulation of oxidized/inactivated DnaK might explain why the CIpP mutant was unable to properly synthesize DNA and proteins, and why it exhibited an aberrant cell morphology. Even though CIpP plays a minor role in the virulence of S. agalactiae in a murine infection model, the data presented here point to the importance of CIpP in oxidative stress defence in preventing heat-induced cell alterations. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty-five per cent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes, 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity. [ABSTRACT FROM AUTHOR]

Summary The dlt operon of Gram-positive bacteria comprises four genes (dltA , dltB , dltC and dltD ) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA ) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50 ) and, in vitro , the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article fluoroquinolone-resistant group B streptococci in acute exacerbation of chronic bronchitis.

Replying to: W. Balemans et al. 462, 10.1038/nature08668 (2009)Our studies led us to conclude that growth of major Gram-positive pathogens, including Staphylococcus aureus, is not inhibited by FASII-targeted antibiotics in septicaemic infection, owing to compensation by serum fatty acids. The comments of Balemans et al. challenge the generality of our results, mainly on the basis of their own work, which is aimed at developing FabI inhibitors for treatment of S. aureus infections. Their allusion to the documented use of FASII inhibitors to treat mycobacterial infections is misleading. Mycobacteria were not considered in our study, because (1) their main route of pathogenesis is not sepsis, and (2) they require mycolic acids for normal growth, which are lacking in serum. The results we present here further reinforce the conclusions of our article. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis. A major virulence factor is a pigmented beta-haemolytic/cyto-lysin (β-h/c) toxin with an ornithine rhamnolipid structure. We initially observed that absence of MprF enzyme altered pigmentation and haemolytic activity in GBS. Next, we showed that MprF-dependent lipid lysination contributes to the retention of the ornithine rhamnolipid within GBS membrane. Furthermore, cationic lipidation by MprF altered membrane properties contributing to resistance to the cyclic lipopeptide daptomycin and to acidic pH. This study highlights the importance of cationic lipids in cell envelope homeostasis and in modulating β-h/c activity., Competing Interests: Declaration of competing interest We declare that there is no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The United Nations Inter-Agency Group for Child Mortality Estimation (UNIGME) estimates that every year 2.5 million neonates die in their first month of life, accounting for nearly one-half of deaths in children under 5 years of age. Neonatal sepsis is the third leading cause of neonatal mortality. The worldwide burden of bacterial sepsis is expected to increase in the next decades due to the lack of effective molecular therapies to replace the administration of antibiotics whose efficacy is compromised by the emergence of resistant strains. In addition, prolonged exposure to antibiotics can have negative effects by increasing the risk of infection by other organisms. With the global burden of sepsis increasing and no vaccine nor other therapeutic approaches proved efficient, the World Health Organization (WHO) stresses the need for new therapeutic targets for sepsis treatment and infection prevention (WHO, A73/32). In response to this unresolved clinical issue, the P2X7 receptor (P2X7R), a key component of the inflammatory cascade, has emerged as a potential target for treating inflammatory/infection diseases. Indeed numerous studies have demonstrated the relevance of the purinergic system as a pharmacological target in addressing immune-mediated inflammatory diseases by regulating immunity, inflammation, and organ function. In this review, we analyze key features of sepsis immunopathophysiology focusing in neonatal sepsis and on how the immunomodulatory role of P2X7R could be a potential pharmacological target for reducing the burden of neonatal sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is not known whether specific mechanisms constrain the emergence of resistance. In this study, we first report ß-lactam tolerance due to the inactivation of the c-di-AMP phosphodiesterase GdpP. Mechanistically, tolerance depends on antagonistic regulation by the repressor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility through the BusAB osmolyte transporter and the AmaP/Asp23/GlsB cell envelope stress complex. The BusR transcriptional response is synergistic with the simultaneous allosteric inhibition of potassium and osmolyte transporters by c-di-AMP, which individually contribute to low-level ß-lactam tolerance. Genome-wide transposon mutagenesis confirms the role of GdpP and highlights functional interactions between a lysozyme-like hydrolase, the KhpAB RNA chaperone and the protein S immunomodulator in the response of GBS to ß-lactam. Overall, we demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response at the transcriptional and post-translational levels to cell wall weakening caused by ß-lactam activity, and reveal additional mechanisms that could foster resistance., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Streptococcus gallolyticus sp. gallolyticus (SGG) is a gut pathobiont involved in the development of colorectal cancer (CRC). To decipher SGG contribution in tumor initiation and/or acceleration respectively, a global transcriptome was performed in human normal colonic cells (FHC) and in human tumoral colonic cells (HT29). To identify SGG-specific alterations, we chose the phylogenetically closest relative, Streptococcus gallolyticus subsp. macedonicus (SGM) as control bacterium. We show that SGM, a bacterium generally considered as safe, did not induce any transcriptional changes on the two human colonic cells. The transcriptional reprogramming induced by SGG in normal FHC and tumoral HT29 cells was significantly different, although most of the genes up- and down-regulated were associated with cancer disease. Top up-regulated genes related to cancer were: (i) IL-20, CLK1, SORBS2, ERG1, PIM1, SNORD3A for normal FHC cells and (ii) TSLP, BHLHA15, LAMP3, ZNF27B, KRT17, ATF3 for cancerous HT29 cells. The total number of altered genes were much higher in cancerous than in normal colonic cells (2,090 vs 128 genes being affected, respectively). Gene set enrichment analysis reveals that SGG-induced strong ER- (endoplasmic reticulum) stress and UPR- (unfolded protein response) activation in colonic epithelial cells. Our results suggest that SGG induces a pro-tumoral shift in human colonic cells particularly in transformed cells potentially accelerating tumor development in the colon., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pasquereau-Kotula et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways., (© 2023. Springer Nature Limited.)

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&#95;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.)

Streptococcus gallolyticus subsp. gallolyticus ( SGG ) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed to characterize the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives of the pathogen. Importantly, we demonstrated that gallocin A peptides can insert themselves into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon, gip , is necessary and sufficient to confer immunity to gallocin A. Structural modeling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide-reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired the ability of gallocin A to kill Enterococcus faecalis. Structural modeling of GIP revealed a hairpin-like structure strongly resembling those of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus ( SGG ), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and is often associated with colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide SGG with an ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (1). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin, recently renamed gallocin A following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and kill phylogenetically closely related bacteria such as most streptococci, lactococci, and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and have very few charged amino acids compared to well-known class IIb bacteriocins. In silico modeling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide forms a hairpin-like structure similar to GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin which displays disulfide bridges important for its structure and activity and might be the founding member of a subtype of class IIb bacteriocins.

Group B Streptococcus (GBS) remains a major neonatal life-threatening pathogen. We initially identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a promising vaccine candidate against GBS. Since GAPDH is highly conserved, we investigate whether GBS GAPDH maternal vaccination interferes with the intestinal colonization of the offspring and the development of its mucosal immune system and central nervous system. An altered gut microbiome with increased Proteobacteria is observed in pups born from vaccinated dams during early life. These pups present decreased relative expression of IL-1β, IL-17A, RegIIIγ and MUC2 in the distal colon. They also display increased CD11b, F4/80 and MHC class II expression on microglia in early life and marked reduction of Ly6C + cells and neutrophils. Importantly, male mice born from vaccinated mothers present behavioral abnormalities during adulthood, including decreased exploratory behavior, a subtle anxious-like phenotype and global alterations in spatial learning and memory strategies, and higher sensitivity to a stressful stimulus. Our study highlights the danger of using ubiquitous antigens in maternal human vaccines against neonatal pathogens., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Streptococcus gallolyticus subspecies gallolyticus ( SGG ) is an opportunistic pathogen causing invasive infections in the elderly often associated with colon neoplasia. The prevalence of SGG in the stools of patients with normal colonoscopy (control) was compared with patients with colorectal adenomas (CRA) or with carcinomas (CRC) from stages I to IV. The presence of the pk s island encoding colibactin as well as other CRC-associated bacteria such as toxicogenic Bacteroides fragilis , Fusobacterium nucleatum , and Parvimonas micra was also investigated., Patients and Methods: Fecal samples collected in France between 2011 and 2016 from patients with normal colonoscopy ( n = 25), adenoma ( n = 23), or colorectal cancer at different stages ( n = 81) were tested by PCR for the presence of SGG , B. fragilis , F. nucleatum , P. micra , and the pks island. Relative quantification of SGG , F. nucleatum , and P. micra in stools was performed by qPCR., Results: SGG prevalence was significantly increased in the CRC group. Our results also revealed i) a strong and significant increase of toxinogenic B. fragilis in patients with early-stage adenoma and of pks island at late-stage CRC and ii) increased levels of F. nucleatum and P. micra in the stools of CRC patients. Furthermore, the simultaneous detection of these five bacterial markers was only found in CRC patients., Conclusions: Our results indicate that the prevalence or relative levels of CRC-associated bacteria vary during CRC development. Among them, B. fragilis ( bft +) was singled out as the sole pathobiont detected at the early adenoma stage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2022 Périchon, Lichtl-Häfele, Bergsten, Delage, Trieu-Cuot, Sansonetti, Sobhani and Dramsi.)

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.

Maternal group B Streptococcus (GBS) colonization is a major risk factor for neonatal GBS infection. However, data on GBS are scarce in low- and middle-income countries. Using sociodemographic data and vaginal swabs collected from an international cohort of mothers and newborns, this study aimed to estimate the prevalence of GBS colonization among pregnant women in Madagascar (n = 1,603) and Senegal (n = 616). The prevalence was 5.0% (95% CI, 3.9-6.1) and 16.1% (95% CI, 13.1-19.0) in Madagascar and Senegal, respectively. No factors among sociodemographic characteristics, living conditions, and obstetric history were found to be associated independently with GBS colonization in both countries. This community-based study provides one of the first estimates of maternal GBS colonization among pregnant women from Madagascar and Senegal.

In a 1945 Nobel Lecture, Sir Alexander Fleming warned against the overuse of antibiotics, particularly in response to public pressure. In the subsequent decades, evidence has shown that bacteria can become resistant to almost any available molecule. One key question is how the emergence and dissemination of resistant bacteria or resistance genes can be delayed. Although some clinicians remain sceptical, in this Personal View, we argue that the prescription of fewer antibiotics and shorter treatment duration is just as effective as longer regimens that remain the current guideline. Additionally, we discuss the fact that shorter antibiotic treatments exert less selective pressure on microorganisms, preventing the development of resistance. By contrast, longer treatments associated with a strong selective pressure favour the emergence of resistant clones within commensal organisms. We also emphasise that more studies are needed to identify the optimal duration of antibiotic therapy for common infections, which is important for making changes to the current guidelines, and to identify clinical biomarkers to guide antibiotic treatment in both hospital and ambulatory settings., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment. IMPORTANCE Staphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections., (Copyright © 2021 Pathania et al.)

Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO 2 to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo -like conditions. IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus , formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions., (Copyright © 2021 Proutière et al.)

Streptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis. We report that S. gallolyticus UCN34 adheres and crosses epithelial monolayers in a Pil3 dependent manner. Confocal images revealed a paracellular passage. Both the Δpil3 mutant and the Pil3+ overexpressing variant were unable to cross Caco-2 and T84 barriers. However, combining live Δpil3 mutant with fixed Pil3+ variant in a 9:1 ratio allowed efficient translocation of the Δpil3 mutant. These results demonstrate that heterogeneous expression of Pil3 plays a key role for UCN34 translocation across the intestinal barrier. Through this skilful strategy, S. gallolyticus probably evade host immune responses., Competing Interests: Declaration of Competing Interest We herein declare that all authors have seen and approved the content of this manuscript and contributed significantly to this work. None of the authors have a financial, personal, or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

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.)

Group B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.

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.

Cyclic di-AMP (c-di-AMP) is a bacterial signaling nucleotide synthesized by several human pathogens. This widespread and specific bacterial product is recognized by infected host cells to trigger an innate immune response. Detection of c-di-AMP in the host cytosol leads primarily to the induction of type I interferon via the STING-cGAS signaling axis, while being also entangled in the activation of the NF-κB pathway. During their long-standing interaction, host and pathogens have co-evolved to control c-di-AMP activation of innate immunity. On the bacterial side, the quantity of c-di-AMP released inside cells allows to manipulate the host response to exacerbate infection by avoiding immune recognition or, at the opposite, by overloading the STING-cGAS pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Colonization by Streptococcus gallolyticus subsp. gallolyticus (SGG) is strongly associated with the occurrence of colorectal cancer (CRC). However, the factors leading to its successful colonization are unknown, and whether SGG influences the oncogenic process or benefits from the tumor-prone environment to prevail remains an open question. Here, we elucidate crucial steps that explain how CRC favors SGG colonization. By using mice genetically prone to CRC, we show that SGG colonization is 1,000-fold higher in tumor-bearing mice than in normal mice. This selective advantage occurs at the expense of resident intestinal enterococci. An SGG-specific locus encoding a bacteriocin ("gallocin") is shown to kill enterococci in vitro. Importantly, bile acids strongly enhance this bacteriocin activity in vivo, leading to greater SGG colonization. Constitutive activation of the Wnt pathway, one of the earliest signaling alterations in CRC, and the decreased expression of the bile acid apical transporter gene Slc10A2 , as an effect of the Apc founding mutation, may thereby sustain intestinal colonization by SGG. We conclude that CRC-specific conditions promote SGG colonization of the gut by replacing commensal enterococci in their niche., Competing Interests: The authors declare no conflict of interest.

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.)

Streptococcus gallolyticus is a commensal bacterium responsible for infectious endocarditis in the elderly, which has frequently been associated with colonic carcinoma. Whether this species is a cause or a consequence of colorectal cancer remains unknown. We recently demonstrated that S. gallolyticus Pil3 pilus is required for adhesion to colonic mucus and for colonization of mouse distal colon. We show here that Pil3 pilus binds equally well to human colonic mucins derived from HT29-MTX cells and to human stomach mucins from healthy donors. In addition, we have found that Pil3 also binds to human fibrinogen, which expands the repertoire of Pil3 host ligands.

The bacterial pathway for fatty acid biosynthesis, FASII, is a target for development of new anti-staphylococcal drugs. This strategy is based on previous reports indicating that self-synthesized fatty acids appear to be indispensable for Staphylococcus aureus growth and virulence, although other bacteria can use exogenous fatty acids to compensate FASII inhibition. Here we report that staphylococci can become resistant to the FASII-targeted inhibitor triclosan via high frequency mutations in fabD, one of the FASII genes. The fabD mutants can be conditional for FASII and not require exogenous fatty acids for normal growth, and can use diverse fatty acid combinations (including host fatty acids) when FASII is blocked. These mutants show cross-resistance to inhibitors of other FASII enzymes and are infectious in mice. Clinical isolates bearing fabD polymorphisms also bypass FASII inhibition. We propose that fatty acid-rich environments within the host, in the presence of FASII inhibitors, might favour the emergence of staphylococcal strains displaying resistance to multiple FASII inhibitors.

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.)