An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

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

Interaction with human plasminogen system turns on proteolytic activity in Streptococcus agalactiae and enhances its virulence in a mouse model

Interactions of several microbial pathogens with the plasminogen system increase their invasive potential. In this study, we show that Streptococcus agalactiae binds human plasminogen which can be subsequently activated to plasmin, thus generating a proteolytic bacterium. S. agalactiae binds plasminogen via the direct pathway, using plasminogen receptors, and via the indirect pathway through fibrinogen receptors. The glyceraldehyde-3-phosphate dehydrogenase is one If the S. agalactiae proteins that bind plasminogen. Presence of exogenous activators such as uPA and tPA are required to activate bound plasminogen. Results from competitive inhibition assays indicate that binding is partially mediated through the lysine binding sites of plasminogen. Following plasminogen binding and activation, S. agalactiae is able to degrade in vitro fibronectin, one of the host extracellular matrix proteins. Moreover, incubation of S. agalactiae with either plasminogen alone, or plasminogen plus fibrinogen, in the presence of tPA enhanced its virulence in C57BL/6 mice, suggesting that acquisition of plasmin-like activity by the bacteria increase their invasiveness

Interaction with human plasminogen system turns on proteolytic activity in Streptococcus agalactiae and enhances its virulence in a mouse model

Interactions of several microbial pathogens with the plasminogen system increase their invasive potential. In this study, we show that Streptococcus agalactiae binds human plasminogen which can be subsequently activated to plasmin, thus generating a proteolytic bacterium. S. agalactiae binds plasminogen via the direct pathway, using plasminogen receptors, and via the indirect pathway through fibrinogen receptors. The glyceraldehyde-3-phosphate dehydrogenase is one If the S. agalactiae proteins that bind plasminogen. Presence of exogenous activators such as uPA and tPA are required to activate bound plasminogen. Results from competitive inhibition assays indicate that binding is partially mediated through the lysine binding sites of plasminogen. Following plasminogen binding and activation, S. agalactiae is able to degrade in vitro fibronectin, one of the host extracellular matrix proteins. Moreover, incubation of S. agalactiae with either plasminogen alone, or plasminogen plus fibrinogen, in the presence of tPA enhanced its virulence in C57BL/6 mice, suggesting that acquisition of plasmin-like activity by the bacteria increase their invasiveness

O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry.

International audienceSerine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein

The Abi-domain protein Abx1 interacts with the CovS histidine kinase to control virulence gene expression in group B Streptococcus

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

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

Lethal meningitis triggered by the hypervirulent group B streptococcus clone ST-17 is mediated by a novel surface protein called HvgA

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

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. \ua9 2014 Macmillan Publishers Limited. All rights reserved

Group B Streptococcus GAPDH Is Released upon Cell Lysis, Associates with Bacterial Surface, and Induces Apoptosis in Murine Macrophages

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages