Metabolism of Poly-ß1,4-N-Acetylglucosamine Substrates and Importation of N-Acetylglucosamine and Glucosamine by Enterococcus faecalis.

The ability of Enterococcus faecalis to use a variety of carbon sources enables colonization at various anatomic sites within a mammalian host. N-Acetylglucosamine (GlcNAc) is one of the most abundant natural sugars and provides bacteria with a source of carbon and nitrogen when metabolized. N-Acetylglucosamine is also a component of bacterial peptidoglycan, further highlighting the significance of N-acetylglucosamine utilization. In this study, we show that CcpA-regulated enzymes are required for growth on the poly-ß1,4-linked GlcNAc substrate, chitopentaose (ß1,4-linked GlcNAc5). We also show that EF0114 (EndoE) is required for growth on chitobiose (ß1,4-linked GlcNAc2) and that the GH20 domain of EndoE is required for the conversion of GlcNAc2 to N-acetylglucosamine. GlcNAc is transported into the cell via two separate phosphotransferase system (PTS) complexes, either the PTS IICBA encoded by ef1516 (nagE) or the Mpt glucose/mannose permease complex (MptBACD). The Mpt PTS is also the primary glucosamine transporter. In order for N-acetylglucosamine to be utilized as a carbon source, phosphorylated N-acetylglucosamine (GlcNAc-6-P) must be deacetylated, and here, we show that this activity is mediated by EF1317 (an N-acetylglucosamine-6-phosphate deacetylase; NagA homolog), as a deletion of ef1317 is unable to grow on GlcNAc as the carbon source. Deamination of glucosamine to fructose-6-phosphate is required for entry into glycolysis, and we show that growth on glucosamine is dependent on EF0466 (a glucosamine-6-phosphate deaminase; NagB homolog). Collectively, our data highlight the chitinolytic machinery required for breaking down exogenous chitinous substrates, as well as the uptake and cytosolic enzymes needed for metabolizing N-acetylglucosamine. IMPORTANCE Enterococcus faecalis causes life-threatening health care-associated infections in part due to its intrinsic and acquired antibiotic resistance, its ability to form biofilms, and its nutrient versatility. Alternative nutrient acquisition systems are key factors that contribute to enterococcal colonization at biologically unique host anatomic sites. Although E. faecalis can metabolize an array of carbon sources, little is known of how this bacterium acquires these secondary nutrient sources in mammalian hosts. Our research identifies the glycosidase machinery required for degrading exogenous chitinous substrates into N-acetylglucosamine monomers for transport and metabolism of one of the most abundant naturally occurring sugars, N-acetylglucosamine. Disrupting the function of this N-acetylglucosamine acquisition pathway may lead to new treatments against multidrug-resistant enterococcal infections.

SigV Mediates Lysozyme Resistance in Enterococcus faecalis via RsiV and PgdA.

Enterococcus faecalis is a gut commensal but transitions to a pathogenic state as a consequence of intestinal dysbiosis and/or the presence of indwelling medical devices, causing a wide range of infections. One of the unique features of E. faecalis is its ability to display high level resistance to lysozyme, an important host defense of the innate immune response. Lysozyme resistance in E. faecalis is known to be mediated by the extracytoplasmic function (ECF) sigma factor SigV. PgdA and RsiV expression is directly regulated by SigV, but pgdA and rsiV mutants display nominal changes in lysozyme resistance, suggesting that additional gene products in the SigV regulon contribute to lysozyme resistance. Using transcriptome sequencing (RNA-seq) analysis, we compared the transcriptional profile of the parental strain to that of an isogenic sigV mutant and show that apart from sigV, only rsiV and pgdA expression was induced upon lysozyme exposure. The combined deletion mutant of both rsiV and pgdA rendered E. faecalis sensitive to lysozyme at a level comparable to that of the sigV mutant, highlighting the limited SigV regulon. Several additional genes were also induced upon lysozyme exposure, but in a SigV-independent fashion. Overexpression of pgdA from a SigV-independent promoter restored lysozyme resistance in a sigV deletion mutant and also induced cell chaining. Overexpression of rsiV from a SigV-independent promoter only partially restored lysozyme resistance in a sigV mutant. Overall, we provide evidence for a simple adaptation to lysozyme stress, in which SigV controls the expression of rsiV and pgdA, and that both gene products contribute to lysozyme resistance. IMPORTANCE Enterococcus faecalis causes health care-associated infections and displays resistance to a variety of antibiotics and molecules of the innate immune system. SigV has been shown to play an important role in enterococcal lysozyme resistance. Even though several proteins have been implicated in enterococcal lysozyme resistance, a complete SigV-dependent regulon has not been functionally characterized as being responsible for the dramatic increase in lysozyme susceptibility displayed by a sigV mutant. Using RNA-seq, we have identified the SigV regulon to be comprised of two gene loci, sigV-rsiV and pgdA. Deletion of both rsiV and pgdA renders E. faecalis susceptible to lysozyme on par with a sigV mutant. We also demonstrate that overproduction of rsiV and pgdA contributes to lysozyme resistance in susceptible strains.

Activity of CcpA-Regulated GH18 Family Glycosyl Hydrolases That Contributes to Nutrient Acquisition and Fitness in Enterococcus faecalis.

The ability of Enterococcus faecalis to colonize host anatomical sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to glycosyl hydrolase family 18 (GH18) (ef0114, ef0361, and ef2863), in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ß-1,4 endo-ß-N-acetylglucosaminidases (ENGases) based on their ß-1,4 endo-N-acetyl-ß-d-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoprotein, an abundant glycan structure on host epithelial surfaces. Here, we show that all three hydrolases are negatively regulated by the transcriptional regulator carbon catabolite protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domains of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose-type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for the deglycosylation of high-mannose-type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and that the glycans removed by EfEndo18A support growth under nutrient-limiting conditions in vitro. In contrast, IgG is representative of a complex-type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for the removal of this glycan decoration. Finally, our data highlight the combined contribution of glycosidases to the virulence of E. faecalis in vivo.

Involvement of Chromosomally Encoded Homologs of the RRNPP Protein Family in Enterococcus faecalis Biofilm Formation and Urinary Tract Infection Pathogenesis.

Enterococcus faecalis is an opportunistic pathogen capable of causing infections, including endocarditis and urinary tract infections (UTI). One of the well-characterized quorum-sensing pathways in E. faecalis involves coordination of the conjugal transfer of pheromone-responsive plasmids by PrgX, a member of the RRNPP protein family. Members of this protein family in various Firmicutes have also been shown to contribute to numerous cellular processes, including sporulation, competence, conjugation, nutrient sensing, biofilm formation, and virulence. As PrgX is a plasmid-encoded RRNPP family member, we surveyed the genome of the multidrug-resistant strain V583 for additional RRNPP homologs using computational searches and refined those identified hits for predicted structural similarities to known RRNPP family members. This led us to investigate the contribution of the chromosomally encoded RRNPP homologs to biofilm processes and pathogenesis in a catheter-associated urinary tract infection (CAUTI) model. In this study, we identified five such homologs and report that 3 of the 5 homologs, EF0073, EF1599, and EF1316, affect biofilm formation as well as outcomes in the CAUTI model.IMPORTANCEEnterococcus faecalis causes health care-associated infections and displays resistance to a variety of broad-spectrum antibiotics by acquisition of resistance traits as well as the ability to form biofilms. Even though a growing number of factors related to biofilm formation have been identified, mechanisms that contribute to biofilm formation are still largely unknown. Members of the RRNPP protein family regulate a diverse set of biological reactions in low-G+C Gram-positive bacteria (Firmicutes). Here, we identify three predicted structural homologs of the RRNPP family, EF0073, EF1599, and EF1316, which affect biofilm formation and CAUTI pathogenesis.

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.

Characterization of natural product inhibitors of quorum sensing in Pseudomonas aeruginosa reveals competitive inhibition of RhlR by ortho-vanillin.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. Ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR.

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 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.

Mechanism of chromosomal transfer of Enterococcus faecalis pathogenicity island, capsule, antimicrobial resistance, and other traits.

The Enterococcus faecalis pathogenicity island (PAI) encodes known virulence traits and >100 additional genes with unknown roles in enterococcal biology. Phage-related integration and excision genes, and direct repeats flanking the island, suggest it moves as an integrative conjugative element (ICE). However, transfer was observed not to require these genes. Transfer only occurred from donors possessing a pheromone responsive-type of conjugative plasmid, and was invariably accompanied by transfer of flanking donor chromosome sequences. Deletion of plasmid transfer functions, including the cis-acting origin of transfer (oriT), abolished movement. In addition to demonstrating PAI movement by a mechanism involving plasmid integration, we observed transfer of a selectable marker placed virtually anywhere on the chromosome. Transfer of this selectable marker was observed to be accompanied by chromosome-chromosome transfer of vancomycin resistance, MLST markers, and capsule genes as well. Plasmid mobilization therefore appears to be a major mechanism for horizontal gene transfer in the evolution of antibiotic resistant E. faecalis strains capable of causing human infection.

Deletion of σ(54) (rpoN) alters the rate of autolysis and biofilm formation in Enterococcus faecalis.

Transcription initiation is a critical step in bacterial gene regulation and is often controlled by transcription regulators. The alternate sigma factor (σ(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 σ(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 σ(54) governs the nature and composition of the biofilm matrix.

The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal.

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 Fsr-dependent 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.

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 (75)Se 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.

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.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.

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 1ß (IL-1ß) 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.

Gelatinase contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis.

The Gram-positive pathogen Enterococcus faecalis is a leading agent of nosocomial infections, including urinary tract infections, surgical site infections, and bacteremia. Among the infections caused by E. faecalis, endocarditis remains a serious clinical manifestation and unique in that it is commonly acquired in a community setting. Infective endocarditis is a complex disease, with many host and microbial components contributing to the formation of bacterial biofilm-like vegetations on the aortic valve and adjacent areas within the heart. In the current study, we compared the pathogenic potential of the vancomycin-resistant E. faecalis V583 and three isogenic protease mutants (ΔgelE, ΔsprE, and ΔgelE ΔsprE mutants) in a rabbit model of enterococcal endocarditis. The bacterial burdens displayed by GelE(-) mutants (ΔgelE and ΔgelE ΔsprE mutants) in the heart were significantly lower than those of V583 or the SprE(-) mutant. Vegetations on the aortic valve infected with GelE(-) mutants (ΔgelE and ΔgelE ΔsprE mutants) also showed a significant increase in deposition of fibrinous matrix layer and increased chemotaxis of inflammatory cells. In support of a role for proteolytic modulation of the immune response to E. faecalis, we also demonstrate that GelE can cleave the anaphylatoxin complement C5a and that this proteolysis leads to decreased neutrophil migration in vitro. In vivo, a decreased heterophil (neutrophil-like cell) migration was observed at tissue sites infected with GelE-producing strains but not at those infected with SprE-producing strains. Taken together, these observations suggest that of the two enterococcal proteases, gelatinase is the principal mediator of pathogenesis in endocarditis.

A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis.

Extracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE-SprE-) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE+SprE+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro- or anti-lytic outcome.

Capsular polysaccharide production in Enterococcus faecalis and contribution of CpsF to capsule serospecificity.

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.

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 its high levels of intrinsic and acquired antibiotic resistance. However, few studies have explored the mechanisms that E. faecalis employs to circumvent the host innate immune response and establish infection. Capsular polysaccharides are important virulence factors that are associated with innate immune evasion. We demonstrate, using cultured macrophages (RAW 264.7), that capsule-producing E. faecalis strains of either serotype C or D are more resistant to complement-mediated opsonophagocytosis than unencapsulated strains. We show that differences in opsonophagocytosis are not due to variations in C3 deposition but are due to the ability of capsule to mask bound C3 from detection on the surface of E. faecalis. Similarly, E. faecalis capsule masks lipoteichoic acid from detection, which correlates with decreased tumor necrosis factor alpha production by cultured macrophages in the presence of encapsulated strains compared to that in the presence of 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.

Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environment.

The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd-Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Enterococcus casseliflavus (10.4%), Enterococcus gallinarum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelatinase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelatinase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4-8 degrees C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies.

Suicide and fratricide in bacterial biofilms.

Bacterial autolysis has recently been identified as a key mechanism that regulates different phases of biofilm development including microcolony formation and dispersal. However such autolytic measures are limited to a subfraction of cells within the entire biofilm population. Here we speculate on the role biofilm heterogeneity plays in limiting autolysis within biofilms and further describe the molecular regulation of suicidal and fratricidal mechanisms in biofilm development of Staphylococcus aureus and Enterococcus faecalis.

Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development.

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.