Eep confers lysozyme resistance to enterococcus faecalis via the activation of the extracytoplasmic function sigma factor SigV

Enterococcus faecalis is a commensal bacterium found in the gastrointestinal tract of most mammals, including humans, and is one of the leading causes of nosocomial infections. One of the hallmarks of E. faecalis pathogenesis is its unusual ability to tolerate high concentrations of lysozyme, which is an important innate immune component of the host. Previous studies have shown that the presence of lysozyme leads to the activation of SigV, an extracytoplasmic function (ECF) sigma factor in E. faecalis, and that the deletion of sigV increases the susceptibility of the bacterium toward lysozyme. Here, we describe the contribution of Eep, a membrane-bound zinc metalloprotease, to the activation of SigV under lysozyme stress by its effects on the stability of the anti-sigma factor RsiV. We demonstrate that the eep mutant phenocopies the sigV mutant in lysozyme, heat, ethanol, and acid stress susceptibility. We also show, using an immunoblot analysis, that in an eep deletion mutant, the anti-sigma factor RsiV is only partially degraded after lysozyme exposure, suggesting that RsiV is processed by unknown protease(s) prior to the action of Eep. An additional observation is that the deletion of rsiV, which results in constitutive SigV expression, leads to chaining of cells, suggesting that SigV might be involved in regulating cell wall-modifying enzymes important in cell wall turnover. We also demonstrate that, in the absence of eep or sigV, enterococci bind significantly more lysozyme, providing a plausible explanation for the increased sensitivity of these mutants toward lysozyme.This work was supported by National Institutes of Health grant 1R01 AI 77782 (L.E.H.)

An ABC Transporter Is Required for Secretion of Peptide Sex Pheromones in Enterococcus faecalis

ABSTRACT Enterococci are leading causes of hospital-acquired infection in the United States and continue to develop resistances to new antibiotics. Many Enterococcus faecalis isolates harbor pheromone-responsive plasmids that mediate horizontal transfer of even large blocks of chromosomal genes, resulting in hospital-adapted strains over a quarter of whose genomes consist of mobile elements. Pheromones to which the donor cells respond derive from lipoprotein signal peptides. Using a novel bacterial killing assay dependent on the presence of sex pheromones, we screened a transposon mutant library for functions that relate to the production and/or activity of the effector pheromone. Here we describe a previously uncharacterized, but well-conserved, ABC transporter that contributes to pheromone production. Using three distinct pheromone-dependent mating systems, we show that mutants defective in expressing this transporter display a 5- to 6-order-of-magnitude reduction in conjugation efficiency. In addition, we demonstrate that the ABC transporter mutant displays an altered biofilm architecture, with a significant reduction in biofilm biomass compared to that of its isogenic parent, suggesting that pheromone activity also influences biofilm development. The conservation of this peptide transporter across the Firmicutes suggests that it may also play an important role in cell-cell communication in other species within this important phylum

Influence of the Alternative Sigma Factor RpoN on Global Gene Expression and Carbon Catabolism in Enterococcus faecalis V583

The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection

Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors

Enterococcus faecalis V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the fsr quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, gelE, sprE, ef1097, ef1351 and ef1352. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding lrgAB, and this induction was found to be lytRS dependent. Drosophila infection was used to discern varying levels of toxicity stemming from mutations in the fsr quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in E. faecalis success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen

Joint sequencing of human and pathogen genomes reveals the genetics of pneumococcal meningitis.

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause life-threatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, identifying variants in CCDC33 associated with susceptibility. Pneumococcal genetic variation explains a large amount of invasive potential (70%), but has no effect on severity. Serotype alone is insufficient to explain invasiveness, suggesting other pneumococcal factors are involved in progression to invasive disease. We identify pneumococcal genes involved in invasiveness including pspC and zmpD, and perform a human-bacteria interaction analysis. These genes are potential candidates for the development of more broadly-acting pneumococcal vaccines

Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains

Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains

<i>Drosophila</i> Host Model Reveals New <i>Enterococcus faecalis</i> Quorum-Sensing Associated Virulence Factors

<div><p><i>Enterococcus faecalis</i> V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the <i>fsr</i> quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, <i>gelE</i>, <i>sprE</i>, <i>ef1097</i>, <i>ef1351</i> and <i>ef1352</i>. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding <i>lrgAB</i>, and this induction was found to be <i>lytRS</i> dependent. <i>Drosophila</i> infection was used to discern varying levels of toxicity stemming from mutations in the <i>fsr</i> quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in <i>E. faecalis</i> success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen.</p></div