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.)

Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion

It has become increasingly difficult to treat infections caused by Enterococcus faecalis due to the high levels of intrinsic and acquired antibiotic resistances. However, few studies have explored the mechanisms that E. faecalis employs to circumvent the host innate immune response and establish infection. Capsule polysaccharides are important virulence factors that are associated with innate immune evasion. We demonstrate that capsule producing E. faecalis strains of either serotype C or D are more resistant to complement-mediated opsonophagocytosis compared to un-encapsulated strains using cultured macrophages (RAW 264.7). We show that differences in opsonophagocytosis are not due to variation in C3 deposition, but due to the ability of capsule to mask bound C3 from detection on the surface of E. faecalis. Similarly, E. faecalis capsule masks detection of lipoteichoic acid which correlates with decreased TNF-α production by cultured macrophages in the presence of encapsulated strains compared to unencapsulated strains. Our studies confirm the important role of the capsule as a virulence factor of E. faecalis, and provide several mechanisms by which the presence of the capsule influences evasion of the innate immune response, and suggest that the capsule could be a potential target for developing alternative therapies to treat E. faecalis infections

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and 1305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.Instituto de Biotecnologia y Biologia Molecula

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

Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial UTIs and pose significant clinical challenges. Enterococcal species are among the predominant causative agents of CAUTIs. However, very little is known about the pathophysiology of Enterococcus-mediated UTIs. We optimized a murine model of foreign body-associated UTI in order to mimic conditions of indwelling catheters in patients. In this model, the presence of a foreign body elicits major histological changes and induces the expression of several proinflammatory cytokines in the bladder. In addition, in contrast to naïve mice, infection of catheter-implanted mice with Enterococcus faecalis induced the specific expression of interleukin 1β (IL-1β) and macrophage inflammatory protein 1α (MIP-1α) in the bladder. These responses resulted in a favorable niche for the development of persistent E. faecalis infections in the murine bladders and kidneys. Furthermore, biofilm formation on the catheter implant in vivo correlated with persistent infections. However, the enterococcal autolytic factors GelE and Atn (also known as AtlA), which are important in biofilm formation in vitro, are dispensable in vivo. In contrast, the housekeeping sortase A (SrtA) is critical for biofilm formation and virulence in CAUTIs. Overall, this murine model represents a significant advance in the understanding of CAUTIs and underscores the importance of urinary catheterization during E. faecalis uropathogenesis. This model is also a valuable tool for the identification of virulence determinants that can serve as potential antimicrobial targets for the treatment of enterococcal infections

Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone

A Selenium-Dependent Xanthine Dehydrogenase Triggers Biofilm Proliferation in Enterococcus faecalis through Oxidant Production

Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with Se-75 is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and 1305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.Instituto de Biotecnologia y Biologia Molecula

The Otterbein Miscellany - May 1967

https://digitalcommons.otterbein.edu/miscellany/1008/thumbnail.jp

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