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. Enterococcus faecalis is a commensal organism present in the mammalian gastrointestinal system (1). Over the past few decades, E. faecalis has arisen as one of the leading causes of nosocomial infection (2). Its role as an opportunistic pathogen is strengthened by the mobile genetic elements it harbors, which are often responsible for conferring resistance to a broad range of antibiotics, including vancomycin (3). In addition, E. faecalis is known to demonstrate a heightened ability to survive in the presence of environmental stress factors, such as increased temperature, acidic pH, and oxidative stress (4). In addition to persistence in the presence of the aforementioned stress factors, previous studies have shown that E. faecalis is also highly resistant to lysozyme (5). This high-level resistance to lysozyme (Ͼ62 mg/ml) is predominantly attributed to the extracytoplasmic function (ECF) sigma factor SigV (5). ECF sigma factors are sequestered by membrane-bound anti-sigma factors and rendered inactive in the absence of a given external stress. Under stress-inducing conditions, the anti-sigma factors are degraded by membrane and cytosolic proteases, leading to the activation of ECF sigma factors in a process referred to as regulated intramembrane proteolysis (RIP) (6).RIP has been shown to play an important role in multiple transmembrane signaling processes associated with increased virulence and environmental fitness (7). In Escherichia ...

Transcription initiation is a critical step in bacterial gene regulation and is often controlled by transcription regulators. The alternate sigma factor ( 54 ) is one such regulator that facilitates activator-dependent transcription initiation and thus modulates the expression of a variety of genes involved in metabolism and pathogenesis in bacteria. This study describes the role of 54 in the nosocomial pathogen Enterococcus faecalis. Biofilm formation is one of the important pathogenic mechanisms of E. faecalis, as it elevates the organism's potential to cause surgical site and urinary tract infections. Lysis of bacterial cells within the population contributes to biofilm formation by providing extracellular DNA (eDNA) as a key component of the biofilm matrix. Deletion of rpoN rendered E. faecalis resistant to autolysis, which in turn impaired eDNA release. Despite the significant reduction in eDNA levels compared to the parental strain, the rpoN mutant formed more robust biofilms as observed using laser scanning confocal microscopy and Comstat analysis, indicating and emphasizing the presence of other matrix components. Initial adherence to a polystyrene surface was also enhanced in the mutant. Proteinase K treatment at early stages of biofilm development significantly reduced the accumulation of biofilm by the rpoN mutant. In conclusion, our data indicate that other factors in addition to eDNA might contribute to the overall composition of the enterococcal biofilm and that the regulatory role of 54 governs the nature and composition of the biofilm matrix.A s opportunistic pathogens, enterococci are the third leading cause of hospital-acquired or associated infections, as they are responsible for 11.2% of surgical site infections (SSI), 14.9% of urinary tract infections (UTI), and 16% of reported bloodstream infections (25). The ability to form a biofilm is an important aspect of the lifestyle of the organism, as biofilm formation is thought to be a property associated with the establishment of SSI and UTI (34), both of which can serve as foci to establish bloodstream infections. Biofilms are aggregates of bacteria that are covered in exoploymer matrix and are more resistant to antibiotics than their planktonic counterparts (15,26). In several bacterial species, nucleic acids, polysaccharides, proteins, and lipids constitute the exopolymer matrix (19). The components of the biofilm matrix form a physical barrier that enhances the inaccessibility of the biofilm cells to antibiotics and the immune system, thereby making the infection difficult to eradicate (33). Extracellular DNA (eDNA) serves as an important biofilm matrix component in several microbial model systems, including but not limited to Neisseria meningitidis, Listeria monocytogenes, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis (2,23,29,32,36,47,48,54). The expression of the two secreted E. faecalis proteases, gelatinase and serine protease, is regulated in a quorum-dependent manner by the Fsr re...

The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsrdependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains.

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. IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.

SUMMARY Streptococcus mutans is implicated in human dental caries, and the carbohydrate metabolism of this organism plays an important role in the formation of this disease. Carbohydrate transport and metabolism are essential for the survival of S. mutans in the oral cavity. It is known that a unique phosphoenolpyruvate-sugar phosphotransferase system PTSBio of S. mutans UA159 is expressed in sucrose-grown biofilms (Mol Oral Microbiol 28: 2013; 114). In this study we analyzed the transcriptional regulation of the operon (OBio) encoding the PTSBio and showed that it was repressed by NigR, a LacI-like transcriptional regulator. Using electromobility shift assay, we described two operators to which NigR bound with different affinities. We also identified the transcriptional start site and showed that one of the operators overlaps with the promoter and presumably represses initiation of transcription. Mutational analyses revealed the key nucleotides in the operators required for high-affinity binding of NigR. PTSBio is expressed in S. mutans biofilms so understanding its regulation may provide improved strategies for caries treatment and prevention.