Search

The commensal bacterium Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity.

Background: Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function., Results: In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model., Conclusions: Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus (A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672-1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.

The Enterococcus faecalis leucine-rich protein ElrA promotes virulence by stimulating bacterial persistence in macrophages and production of the interleukin-6 (IL-6) cytokine. The ElrA protein is encoded within an operon that is poorly expressed under laboratory conditions but induced in vivo. In this study, we identify ef2687 (renamed elrR), which encodes a member of the Rgg (regulator gene for glucosyltransferase) family of putative regulatory proteins. Using quantitative reverse transcription-PCR, translational lacZ fusions, and electrophoretic mobility shift assays, we demonstrate that ElrR positively regulates expression of elrA. These results correlate with the attenuated virulence of the ΔelrR strain in a mouse peritonitis model. Virulence of simple and double elrR and elrA deletion mutants also suggests a remaining ElrR-independent expression of elrA in vivo and additional virulence-related genes controlled by ElrR.

Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.

Peptidylprolyl cis/trans isomerases (PPIases) are enzymes involved in protein folding. Analysis of the genome sequence of Enterococcus faecalis V583 allowed for identification of 3 PPIases carrying genes. ef2898 encodes an intracellular PPIase which was not shown to be important for the E. faecalis stress response or virulence. The other two PPIases, the parvulin family rotamase EF0685 and the cyclophilin family member EF1534, are expected to be surface-exposed proteins. They were shown to be important for virulence and resistance to NaCl. A Δef0685 Δef1534 mutant was also more resistant to oxidative stress, was able to grow under a high manganese concentration, and showed altered resistance to ampicillin and quinolone antibiotics.

Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyA mutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo-in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

Methionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S- and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrA gene and one msrB gene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalis strains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H2O2, and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrA mutant is attenuated in the Galleria mellonella insect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.

Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

The intracellular survival in mouse peritoneal macrophages of 8 Enterococcus faecalis response regulator mutants was tested to assess if the corresponding 2-component signal transduction systems (TCS) are involved in the ability of E. faecalis to survive in macrophages. Three mutants (err04, err05 and err06) are more susceptible than the wild-type JH2-2 strain and 1 is more resistant (err10). Then, characterization of the TCS Err04-Ehk04 and Err06-Ehk06 reveals that the first (homolog of PhoP-PhoR of Bacillus subtilis) is induced in phosphate deprivation conditions, regulates its own expression and plays a role in the expression of pstF encoding a phosphate-binding protein. The Err06-Ehk06 is involved in oxidative stress response. A mutation in the err06 gene increases sensitivity of the bacterium to H(2)O(2). The err06-ehk06 operon is induced by H(2)O(2) stress and controlled by 2 transcriptional start sites, of which 1 is specifically active in oxidative stress conditions. We also demonstrated that the expression of the catalase gene (kat) is partly dependant of the Err06-Ehk06 TCS.

The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H(2)O(2). Exposure to internal generated H(2)O(2) by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H(2)O(2) concentrations have been determined. This showed that the time point of growth arrest of the Deltanpr mutant correlated with the highest H(2)O(2) concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the Deltatpx and the triple mutant showed attenuated virulence in a mouse peritonitis model.

Enterococcus faecalis is an important nosocomial pathogen associated with high morbidity and mortality for patients who are immunocompromised or who have severe underlying diseases. The E. faecalis genome encodes numerous surface-exposed proteins that may be involved in virulence. This work describes the characterization of the first internalin-like protein in E. faecalis, ElrA, belonging to the recently identified WxL family of surface proteins. ElrA contains an N-terminal signal peptide for export, a leucine-rich repeat domain that may interact with host cells, and a C-terminal WxL domain that interacts with the peptidoglycan. Disruption of the elrA gene significantly attenuates bacterial virulence in a mouse peritonitis model. The elrA deletion mutant also displays a defect in infection of host macrophages and a decreased interleukin-6 response in vivo. Finally, elrA expression is induced in vivo. Altogether, these results demonstrate a role for ElrA in the E. faecalis infectious process in vivo and suggest that this surface protein may contribute to E. faecalis virulence by stimulating the host inflammatory response.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (P < 0.05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

In order to identify regulators of the oxidative stress response in Enterococcus faecalis, an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydroperoxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis.

The intracellular survival in mouse peritoneal macrophages of 8 Enterococcus faecalis response regulator mutants was tested to assess if the corresponding 2-component signal transduction systems (TCS) are involved in the ability of E. faecalis to survive in macrophages. Three mutants (err04, err05 and err06) are more susceptible than the wild-type JH2-2 strain and 1 is more resistant (err10). Then, characterization of the TCS Err04-Ehk04 and Err06-Ehk06 reveals that the first (homolog of PhoP-PhoR of Bacillus subtilis) is induced in phosphate deprivation conditions, regulates its own expression and plays a role in the expression of pstF encoding a phosphate-binding protein. The Err06-Ehk06 is involved in oxidative stress response. A mutation in the err06 gene increases sensitivity of the bacterium to H2O2. The err06-ehk06 operon is induced by H2O2 stress and controlled by 2 transcriptional start sites, of which 1 is specifically active in oxidative stress conditions. We also demonstrated that the expression of the catalase gene (kat) is partly dependant of the Err06-Ehk06 TCS. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

E. faecalis and E. faecium cause urinary tract infections highly resistant to therapies due to a protective extracellular matrix. To exploit a new strategy able to treat infections without increasing antibiotic doses, we used enzymes targeting specific biofilm matrix components in combination with Vancomycin. We investigated the activity of Vancomycin combined with two matrix-degrading enzymes, Alginate Lyase (AlgL) and Deoxyribonuclease I (DNase I) against in vitro biofilm of E. faecalis and E. faecium clinical isolates. The heterogeneity of matrix composition leads to defined physiological responses of biofilm communities to their environment: we demonstrated that the use of DNase I and AlgL enzymes affects biofilm structure, cell viability and reduces MBEC values of Vancomycin in E. faecalis and E. faecium, respectively. [ABSTRACT FROM AUTHOR]