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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.)

Streptococcus gallolyticus is an increasing cause of bacteremia and infective endocarditis in the elderly. Several epidemiological studies have associated the presence of this bacterium with colorectal cancer. We have studied the interaction of S. gallolyticus with human colonic cells. S. gallolyticus strain UCN34, adhered better to mucus-producing cells such as HT-29-MTX than to the parental HT-29 cells. Attachment to colonic mucus is dependent on the pil3 pilus operon, which is heterogeneously expressed in the wild-type UCN34 population. We constructed a pil3 deletion mutant in a Pil3 overexpressing variant (Pil3+) and were able to demonstrate the role of Pil3 pilus in binding to colonic mucus. Importantly, we showed that pil3 deletion mutant was unable to colonize mice colon as compared to the isogenic Pil3+ variant. Our findings establish for the first time a murine model of intestinal colonization by S. gallolyticus., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

Summary: 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 αv 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. The mechanisms by which the bacterial pathogen group B streptococcus (GBS) interacts with host cells are incompletely understood. We show here that the extracellular matrix protein vitronectin acts as a bridge between the bacterial cell wall protein PbsP and integrins present on the host cell membrane. These interactions enable GBS to adhere to epithelial cells and invade them. Drugs or antibodies capable of blocking these interactions may be useful to control infections by GBS. [ABSTRACT FROM AUTHOR]

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. [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]

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 &y& Elsevier]

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]