Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum.
Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.
SummaryGroup B streptococci (GBS) express a b-haemolysin/ cytolysin that contributes to disease pathogenesis. We report an independent discovery and extension of a genetic locus encoding the GBS b-haemolysin/cytolysin activity. A plasmid library of GBS chromosomal DNA was cloned into Escherichia coli, and a transformant was identified as b-haemolytic on blood agar. The purified plasmid contained a 4046 bp insert of GBS DNA encoding two complete open reading frames (ORFs). A partial upstream ORF (cylB) and the first complete ORF (cylE) represent the 3 H end of a newly reported genetic locus (cyl) required for GBS haemolysin/cytolysin activity. ORF cylE is predicted to encode a 78.3 kDa protein without GenBank homologies. The GBS DNA fragment also includes a previously unreported ORF, cylF, with homology to bacterial aminomethyltransferases, and the 5 H end of cylH, with homology to 3-ketoacyl-ACP synthases. Southern analysis demonstrated that the cyl locus was conserved among GBS of all common serotypes. Targeted plasmid integrational mutagenesis was used to disrupt cylB, cylE, cylF and cylH in three wild-type GBS strains representing serotypes Ia, III and V. Targeted integrations in cylB, cylF and cylH retaining wild-type haemolytic activity were identified in all strains. In contrast, targeted integrations in cylE were invariably non-haemolytic and non-cytolytic, a finding confirmed by in frame allelic exchange of the cylE gene. The haemolytic/cytolytic activity of the cylE allelic exchange mutants could be restored by reintroduction of cylE on a plasmid vector. Inducible expression of cylE, cylF and cylEF demonstrated that it is CylE that confers haemolytic activity in E. coli. We conclude that cylE probably represents the structural gene for the GBS haemolysin/cytolysin, a novel bacterial toxin.
Recently, cholesterol was identified as a physiologically important nutrient for Mycobacterium tuberculosis survival in chronically infected mice. However, it remained unclear precisely when cholesterol is available to the bacterium and what additional bacterial functions are required for its metabolism. Here, we show that the igr locus, which we previously found to be essential for intracellular growth and virulence of M. tuberculosis, is required for cholesterol metabolism. While igr-deficient strains grow identically to the wild type in the presence of short-and long-chain fatty acids, the growth of these bacteria is completely inhibited in the presence of cholesterol. Interestingly, this mutant is still able to respire under cholesterol-dependent growth inhibition, suggesting that the bacteria can metabolize other carbon sources during cholesterol toxicity. Consistent with this hypothesis, we found that the growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as mutation of the mce4 sterol uptake system partially suppresses this effect. In addition, the ⌬igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout infection.
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