Utilization of glycoprotein-derived N-acetylglucosamine-L-asparagine during Enterococcus faecalis infection depends on catabolic and transport enzymes of the glycosylasparaginase locus.

Enterococcus faecalis is a Gram-positive clinical pathogen causing severe infections. Its survival during infection depends on its ability to utilize host-derived metabolites, such as protein-deglycosylation products. We have identified in E. faecalis OG1RF a locus (ega) involved in the catabolism of the glycoamino acid N-acetylglucosamine-L-asparagine. This locus is separated into two transcription units, genes egaRP and egaGBCD1D2, respectively. RT-qPCR experiments revealed that the expression of the ega locus is regulated by the transcriptional repressor EgaR. Electromobility shift assays evidenced that N-acetylglucosamine-L-asparagine interacts directly with the EgaR protein, which leads to the transcription of the ega genes. Growth studies with egaG, egaB and egaC mutants confirmed that the encoded proteins are necessary for N-acetylglucosamine-L-asparagine catabolism. This glycoamino acid is transported and phosphorylated by a specific phosphotransferase system EIIABC components (OG1RF_10751, EgaB, EgaC) and subsequently hydrolyzed by the glycosylasparaginase EgaG, which generates aspartate and 6-P-N-acetyl-ß-d-glucosaminylamine. The latter can be used as a fermentable carbon source by E. faecalis. Moreover, Galleria mellonella larvae had a significantly higher survival rate when infected with ega mutants compared to the wild-type strain, suggesting that the loss of N-acetylglucosamine-L-asparagine utilization affects enterococcal virulence.

The enigmatic physiological roles of AhpCF, Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium.

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H2O2) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H2O2 which avoids accumulation of the peroxide to lethal concentrations.

Characterisation of the manganese superoxide dismutase of Enterococcus faecium.

The manganese superoxide dismutase (SodA) of E. faecium strain AUS0004 has been characterised. It is most closely related to Enterococcus hirae, Enterococcus durans, Enterococcus villorium, and Enterococcus mundtii with 100%, 91,55%, 90,85%, and 90,58% homology, respectively, but more distant from SodA of E. faecalis (81.68%). A sodA deletion mutant has been constructed. Compared to the parental strain, the ΔsodA mutant was affected in aerobic growth and more sensitive to hydrogen peroxide (H2O2), cumene hydroperoxide (CuOOH), and the superoxide anion (O2•-) generator menadione. The E. faecium strain AUS0004 is part of those bacteria accumulating H2O2 to high concentrations (around 5 mM) starting from late exponential growth phase. Accumulation of the peroxide was around 25% less in the mutant suggesting that this part of H2O2 is due to the dismutation of O2•- by SodA. The sodA gene of E. faecium AUS0004 was induced by oxygen, peroxides and menadione but the corresponding regulator remains hitherto unknown. Finally, we showed that SodA activity is important for virulence in the Galleria mellonella model.

Inhibition of d-alanylation of teichoic acids overcomes resistance of methicillin-resistant Staphylococcus aureus.

BACKGROUND: MRSA are high-priority multidrug-resistant pathogens. Although there are still some antibiotics active against MRSA, continuous efforts to discover new antibiotics and treatment strategies are needed because resistance to these new drugs has already been reported. OBJECTIVES: Here we explore if d-alanylation of teichoic acids (TAs) mediated by the dlt operon gene products might be a druggable target to overcome ß-lactam-resistance of MRSA. METHODS: MICs and bactericidal effects of several ß-lactam antibiotics were monitored in a panel of clinical MRSA strains with genetic or chemically induced deficiency in d-alanylation of TAs. Efficiency of the chemical inhibitor to rescue MRSA-infected larvae of Galleria mellonella as well as its ability to prevent or eradicate biofilms of S. aureus were analysed. RESULTS: Genetic inactivation of the Dlt system or its chemical inhibition re-sensitizes MRSA to ß-lactams. Among the 13 strains, the most pronounced effect was obtained using the inhibitor with imipenem, reducing the median MIC from 16 to 0.25 mg/L. This combination was also bactericidal in some strains and significantly protected G. mellonella larvae from lethal MRSA infections. Finally, inactivation of d-alanylation potentiated the effect of imipenem on inhibition and/or eradication of biofilm. CONCLUSIONS: Our combined results show that highly efficient inhibitors of d-alanylation of TAs targeting enzymes of the Dlt system should be promising therapeutic adjuvants, especially in combination with carbapenems, for restoring the therapeutic efficacy of this class of antibiotics against MRSA.

Analysis of glycerol and dihydroxyacetone metabolism in Enterococcus faecium.

Glycerol (Gly) can be dissimilated by two pathways in bacteria. Either this sugar alcohol is first oxidized to dihydroxyacetone (DHA) and then phosphorylated or it is first phosphorylated to glycerol-3-phosphate (GlyP) followed by oxidation. Oxidation of GlyP can be achieved by NAD-dependent dehydrogenases or by a GlyP oxidase. In both cases, dihydroxyacetone phosphate is the product. Genomic analysis showed that Enterococcus faecium harbors numerous genes annotated to encode activities for the two pathways. However, our physiological analyses of growth on glycerol showed that dissimilation is limited to aerobic conditions and that despite the presence of genes encoding presumed GlyP dehydrogenases, the GlyP oxidase is essential in this process. Although E. faecium contains an operon encoding the phosphotransfer protein DhaM and DHA kinase, which are required for DHA phosphorylation, it is unable to grow on DHA. This operon is highly expressed in stationary phase but its physiological role remains unknown. Finally, data obtained from sequencing of a transposon mutant bank of E. faecium grown on BHI revealed that the GlyP dehydrogenases and a major intrinsic family protein have important but hitherto unknown physiological functions.

ß-Lactam Exposure Triggers Reactive Oxygen Species Formation in Enterococcus faecalis via the Respiratory Chain Component DMK.

Whereas the primary actions of ß-lactams are well characterized, their downstream effects are less well understood. Although their targets are extracellular, ß-lactams stimulate respiration in Escherichia coli leading to increased intracellular accumulation of reactive oxygen species (ROS). Here, we show that ß-lactams over a large concentration range trigger a strong increase in ROS production in Enterococcus faecalis under aerobic, but not anaerobic, conditions. Both amoxicillin, to which the bacterium is susceptible, and cefotaxime, to which E. faecalis is resistant, triggers this response. This stimulation of ROS formation depends mainly on demethylmenaquinone (DMK), a component of the E. faecalis respiratory chain, but in contrast to E. coli is observed only in the absence of respiration. Our results suggest that in E. faecalis, ß-lactams increase electron flux through the respiratory chain, thereby stimulating the auto-oxidation of reduced DMK in the absence of respiration, which triggers increased extracellular ROS production.

Genetic and pharmacological inactivation of d-alanylation of teichoic acids sensitizes pathogenic enterococci to ß-lactams.

BACKGROUND: Enterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging. OBJECTIVES: Teichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by d-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections. METHODS: The susceptibility of a d-alanylation-deficient strain of Enterococcus faecalis to ß-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of d-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium. RESULTS: Most cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1 mg/L) and amoxicillin (0.03 mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium. CONCLUSIONS: The combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.

Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

BACKGROUND: Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens. RESULTS: We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens. CONCLUSION: Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins together with polysaccharide antigens in vaccine development against enterococcal infections.

Transcriptomic response of Enterococcus faecalis V583 to low hydrogen peroxide levels.

Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of human and other mammals, but is also increasingly recognized as an opportunistic human pathogen. Oxidative stress is one of the major challenges encountered by enterococci, both in their natural environment and during infection. In this paper, we evaluated the transcriptomic response of E. faecalis to oxidative stress, and showed that transcript abundance was reduced for 93 genes and increased for 39 genes during growth in medium containing 1.75 mM H2O2. The presence of hydrogen peroxide affected several metabolic pathways, including a large decrease in ethanolamine utilization and methylglyoxal metabolism, and an increase in transcript abundance for several transport systems. In particular, four operons encoding iron transporters appeared highly induced. By contrast, in our experimental conditions, the expression of most of the genes known to be involved in the enterococcal response to oxidative stress, did not appear significantly altered.

Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections.

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.

Proteome phenotyping of ΔrelA mutants in Enterococcus faecalis V583.

The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583. A 2-dimensional electrophoresis proteomic analysis of 2 relA mutants, i.e., ΔrelA carrying a complete deletion of the relA gene, and ΔrelAsp that is deleted from only its 3' extremity, showed that 31 proteins were deregulated in 1 or both of these mutants. Mass spectrometry identification of these proteins showed that 10 are related to translation, including 5 ribosomal proteins, 3 proteins involved in translation elongation, and 2 proteins in tRNA synthesis; 14 proteins are involved in diverse metabolisms and biosynthesis (8 in sugar and energy metabolisms, 2 in fatty acid biosynthesis, 2 in amino acid biosynthesis, and 2 in nucleotide metabolism). Five proteins were relevant to the adaptation to different environmental stresses, i.e., SodA and a Dps family protein, 2 cold-shock domain proteins, and Ef1744, which is a general stress protein that plays an important role in the response to ethanol stress. The potential role of these proteins in the development of stress phenotypes associated with these mutations is discussed.

Involvement of Enterococcus faecalis small RNAs in stress response and virulence.

Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.

CspR, a cold shock RNA-binding protein involved in the long-term survival and the virulence of Enterococcus faecalis.

By coprecipitation, we identified RNA-binding proteins in the Gram-positive opportunistic pathogen Enterococcus faecalis known to be deficient of the RNA chaperone Hfq. In particular, we characterized one belonging to the cold shock protein (Csp) family (Ef2925) renamed CspR for cold shock protein RNA binding. Compared to the wild-type strain, the ΔcspR mutant was less virulent in an insect infection model (Galleria mellonella) and exhibited a decreased persistence in mouse kidneys and a low survival rate in peritoneal macrophages. As expected, we found that the ΔcspR mutant strain was more impaired in its growth than the parental strain under cold conditions and in its long-term survival under nutrient starvation. All these phenotypes were restored after complementation of the ΔcspR mutant. In addition, Western blot analysis showed that CspR was overexpressed under cold shock conditions and in the stationary phase. Since CspR may act as an RNA chaperone, putative targets were identified using a global proteomic approach completed with transcriptomic assays. This study revealed that 19 proteins were differentially expressed in the ΔcspR strain (9 upregulated, 10 downregulated) and that CspR mainly acted at the posttranscriptional level. These data highlight for the first time the role of the RNA-binding protein CspR as a regulator in E. faecalis and its requirement in stress response and virulence in this important human pathogen.

Catabolic pathway of gamma-caprolactone in the biocontrol agent Rhodococcus erythropolis.

Gamma-caprolactone (GCL) is well-known as a food flavor and has been recently described as a biostimulant molecule promoting the growth of bacteria with biocontrol activity against soft-rot pathogens. Among these biocontrol agents, Rhodococcus erythropolis, characterized by a remarkable metabolic versatility, assimilates various γ-butyrolactone molecules with a branched-aliphatic chain, such as GCL. The assimilative pathway of GCL in R. erythropolis was investigated by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis. This analysis suggests the involvement of the lactonase QsdA in ring-opening, a feature confirmed by heterologous expression in Escherichia coli. According to proteome analysis, the open-chain form of GCL was degraded by ß- and ω-oxidation coupled to the Krebs cycle and ß-ketoadipate pathway. Ubiquity of qsdA gene among environmental R. erythropolis isolates was verified by PCR. In addition to a previous N-acyl homoserine lactone catabolic function, QsdA may therefore be involved in an intermediate degradative step of cyclic recalcitrant molecules or in synthesis of flavoring lactones.

The prolipoprotein diacylglyceryl transferase (Lgt) of Enterococcus faecalis contributes to virulence.

Enterococcus faecalis is an opportunistic pathogen responsible for nosocomial infections. Lipoproteins in Gram-positive bacteria are translocated across the plasma membrane and anchored by the fatty acid group. They perform critical roles, with some described as virulence determinants. The aim of this study was to explore the roles of E. faecalis lipoproteins in the stress response and virulence. We constructed a mutant affected in the predicted prolipoprotein diacylglyceryl transferase gene lgt, and examined the role of Lgt in membrane anchoring, growth, the stress response and virulence. Inactivation of lgt enhanced growth in a high concentration of Mn(2+) or under oxidative stress in vitro, and significantly decreased virulence.

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors.

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

Genome-wide identification of small RNAs in the opportunistic pathogen Enterococcus faecalis V583.

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5' and 3' RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis.

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.

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.