The lysozyme-induced peptidoglycan N-acetylglucosamine deacetylase PgdA (EF1843) is required for Enterococcus faecalis virulence.

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia.

OBJECTIVES: A previous study identified an association between high MICs of quaternary ammonium compounds (QACs) and antibiotic resistance. The current aim was to investigate the genetic background of this association. METHODS: Of 153 Escherichia coli clinical strains, seven were selected for their low or high MICs of antibiotics and/or QACs. Integron resistance gene contents were identified by sequencing after PCR amplification. The genes encoding the efflux pump AcrA/TolC and its regulatory regions marA, marO, marR, soxS and rob were sequenced. The gene expression of acrA, tolC, marA, marOR, soxS and rob was assessed by quantitative real-time PCR. MICs in the presence and absence of the efflux pump inhibitor phenyl-arginine-ß-naphthylamide (PAßN) were compared. RESULTS: Of the seven strains, five were resistant to amoxicillin, amoxicillin/clavulanic acid and/or co-trimoxazole (trimethoprim/sulfamethoxazole) and/or had high MICs of ciprofloxacin and QACs. Four of the five harboured a class 1 integron (intI1). In three of these four strains, the presence of dfrA/sul1 and qacEΔ1 gene cassettes correlated with resistance to co-trimoxazole and high MICs of QACs. In all of the five strains, overexpression of tolC, marOR and soxS was always associated with higher MICs of antibiotics and/or QACs. PAßN reduced the MICs of ciprofloxacin and QACs, suggesting that extrusion of ciprofloxacin and QACs from bacteria depends on the AcrAB-TolC system. CONCLUSIONS: To our knowledge, this report is the first to describe dual involvement of the AcrAB-TolC system and class 1 integrons in clinical E. coli strains.

Identification of secreted and surface proteins from Enterococcus faecalis.

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis, the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography - electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

Impaired functions of macrophage from cystic fibrosis patients: CD11b, TLR-5 decrease and sCD14, inflammatory cytokines increase.

BACKGROUND: Early in life, cystic fibrosis (CF) patients are infected with microorganisms. The role of macrophages has largely been underestimated in literature, whereas the focus being mostly on neutrophils and epithelial cells. Macrophages may however play a significant role in the initiating stages of this disease, via an inability to act as a suppressor cell. Yet macrophage dysfunction may be the first step in cascade of events leading to chronic inflammation/infection in CF. Moreover, reports have suggested that CFTR contribute to altered inflammatory response in CF by modification of normal macrophage functions. OBJECTIVES: In order to highlight possible intrinsic macrophage defects due to impaired CFTR, we have studied inflammatory cytokines secretions, recognition of pathogens and phagocytosis in peripheral blood monocyte-derived macrophages from stable adult CF patients and healthy subjects (non-CF). RESULTS: In CF macrophage supernatants, concentrations of sCD14, IL-1ß, IL-6, TNF-α and IL-10 were strongly raised. Furthermore expression of CD11b and TLR-5 were sorely decreased on CF macrophages. Beside, no difference was observed for mCD14, CD16, CD64, TLR-4 and TLR1/TLR-2 expressions. Moreover, a strong inhibition of phagocytosis was observed for CF macrophages. Elsewhere CFTR inhibition in non-CF macrophages also led to alterations of phagocytosis function as well as CD11b expression. CONCLUSIONS: Altogether, these findings demonstrate excessive inflammation in CF macrophages, characterized by overproduction of sCD14 and inflammatory cytokines, with decreased expression of CD11b and TLR-5, and impaired phagocytosis. This leads to altered clearance of pathogens and non-resolution of infection by CF macrophages, thereby inducing an exaggerated pro-inflammatory response.

Construction of a new sensitive molecular tool for the study of gene expression in Enterococcus faecalis.

A promoter-probe vector designated pVEPhoZ-P has been developed to provide a convenient system to analyze transcription activities of promoters. It was constructed on the basis of pVE14218, a plasmid which lacks the replication protein and in which the 5' part of alkaline phosphatase (AP) phoZ gene of Enterococcus faecalis was inserted. The pVEPhoZ vector was used to clone promoters of interest. The resulting promoter-probe vectors were integrated in the phoZ chromosomal locus by homologous recombination. This procedure generates recombinant clones with a single copy of phoZ functional allele placed under the control of the desired promoter. This system was investigated with different promoters of E. faecalis genes, namely those of sigV encoding an ECF sigma factor, croRS encoding a two-component system and dhaK operon. In all cases, expression data obtained previously for the three promoters were properly reported for this new tool. The pVEPhoZ-P promoter-probe vector is easy to use and it showed higher reporter activity in comparison to systems based on ß-galactosidase. Therefore, this system constitutes a useful molecular tool for the study of promoters in E. faecalis or other bacterial species that possess a homologous phoZ gene.

The extracytoplasmic function sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis.

BACKGROUND: Enterococcus faecalis is one of the leading agents of nosocomial infections. To cause diseases, pathogens or opportunistic bacteria have to adapt and survive to the defense systems encountered in the host. One of the most important compounds of the host innate defense response against invading microorganisms is lysozyme. It is found in a wide variety of body fluids, as well as in cells of the innate immune system. Lysozyme could act either as a muramidase and/or as a cationic antimicrobial peptide. Like Staphylococcus aureus, E. faecalis is one of the few bacteria that are completely lysozyme resistant. RESULTS: This study revealed that oatA (O-acetyl transferase) and dlt (D-Alanylation of lipoteicoic acids) genes contribute only partly to the lysozyme resistance of E. faecalis and that a specific transcriptional regulator, the extracytoplasmic function SigV sigma factor plays a key role in this event. Indeed, the sigV single mutant is as sensitive as the oatA/dltA double mutant, and the sigV/oatA/dltA triple mutant displays the highest level of lysozyme sensitivity suggesting synergistic effects of these genes. In S. aureus, mutation of both oatA and dlt genes abolishes completely the lysozyme resistance, whereas this is not the case in E. faecalis. Interestingly SigV does not control neither oatA nor dlt genes. Moreover, the sigV mutants clearly showed a reduced capacity to colonize host tissues, as they are significantly less recovered than the parental JH2-2 strain from organs of mice subjected to intravenous or urinary tract infections. CONCLUSIONS: This work led to the discovery of an original model of lysozyme resistance mechanism which is obviously more complex than those described for other Gram positive pathogens. Moreover, our data provide evidences for a direct link between lysozyme resistance and virulence of E. faecalis.

Ers a Crp/Fnr-like transcriptional regulator of Enterococcus faecalis.

Ers has been identified in a recent study as a protein involved in the pathogenesis and the stress response of the lactic acid bacterium Enterococcus faecalis, an opportunistic pathogen. In the E. faecalis sequenced genome, Ers is annotated as a transcriptional regulator member of the Crp/Fnr family. This protein has been shown to be involved in the oxidative stress response as well as in the survival within macrophages. In the present study, we sum up the characteristics of Ers and provide further evidence that this protein is a member of the PrfA branch of this regulator family. These features emphasize the importance of studying Ers since PrfA is the major regulator of virulence in Listeria monocytogenes. Ers shares common Crp/Fnr family characteristics, including a HTH motif, a cyclic nucleotide binding domain and conserved amino acid residues. Furthermore, a "PrfA-box-like" sequence has been identified in the ers promoter region. A similar sequence is present in the ef0082 promoter, a gene known to be a member of the Ers regulon. Moreover, ers shares the same genetic neighborhood as other PrfA-like proteins, present in Gram positive bacteria. Lastly, by comparison with PrfA, we have identified an amino acid substitution in the Ers sequence. Such a substitution could imply that Ers is in a constitutively active form.