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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 Δeep mutant phenocopies the ΔsigV 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]