RESUMEN
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.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Antibacterianos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus , Ácidos Teicoicos , beta-Lactamas/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enterococcus/efectos de los fármacos , Enterococcus/crecimiento & desarrollo , Ácidos Teicoicos/genética , beta-Lactamas/farmacología , Subfamilia D de Transportadores de Casetes de Unión al ATP/genética , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Enterococcus/genética , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Infecciones por Bacterias Grampositivas/microbiología , Larva/microbiología , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Ácidos Teicoicos/químicaRESUMEN
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-6â³-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6â³-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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Enterococcus faecalis/enzimología , Enterococcus faecalis/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Polisacáridos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Riñón/microbiología , Hígado/microbiología , Maltosa/farmacología , Proteínas de Transporte de Membrana/genética , Ratones , Mutación , Oligosacáridos/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Trisacáridos/farmacologíaRESUMEN
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.
Asunto(s)
Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Transcriptoma , Adaptación Biológica/genética , Transporte Biológico , Relación Dosis-Respuesta a Droga , Enterococcus faecalis/metabolismo , Etanolamina/metabolismo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Anotación de Secuencia Molecular , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Piruvaldehído/metabolismoRESUMEN
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.
Asunto(s)
Enterococcus faecalis/genética , Estrés Fisiológico/genética , Virulencia/genética , Animales , Femenino , Tracto Gastrointestinal/microbiología , Regulación Bacteriana de la Expresión Génica/genética , Infecciones por Bacterias Grampositivas/genética , Infecciones por Bacterias Grampositivas/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/genética , Ligasas/genética , Proteoma/metabolismo , Proteínas Bacterianas/genética , Enterococcus faecalis/enzimología , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Redes y Vías Metabólicas , Mutación , Fenotipo , Biosíntesis de Proteínas , Proteoma/genética , Estrés FisiológicoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas y Péptidos de Choque por Frío/metabolismo , Enterococcus faecalis/fisiología , Enterococcus faecalis/patogenicidad , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas y Péptidos de Choque por Frío/genética , Enterococcus faecalis/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Infecciones por Bacterias Grampositivas/microbiología , Proteína de Factor 1 del Huésped/metabolismo , Riñón/microbiología , Macrófagos Peritoneales/microbiología , Ratones , Viabilidad Microbiana , Mariposas Nocturnas/microbiología , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Estrés Fisiológico/genéticaRESUMEN
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.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Proteínas Bacterianas/metabolismo , Rhodococcus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Electroforesis en Gel Bidimensional , Escherichia coli , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Redes y Vías Metabólicas , Peso Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/enzimología , Rhodococcus/crecimiento & desarrollo , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
Enterococcus faecalis/enzimología , Enterococcus faecalis/patogenicidad , Transferasas/metabolismo , Factores de Virulencia/metabolismo , Animales , Modelos Animales de Enfermedad , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Eliminación de Gen , Prueba de Complementación Genética , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/mortalidad , Larva/microbiología , Lepidópteros , Manganeso/metabolismo , Análisis de Supervivencia , Transferasas/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Enterococcus faecium , Glutatión Peroxidasa , Catalasa/genética , Enterococcus faecium/genética , Peróxidos , Peróxido de Hidrógeno/farmacología , PeroxidasasRESUMEN
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.
Asunto(s)
Dihidroxiacetona/metabolismo , Enterococcus faecium/enzimología , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enterococcus faecium/genética , Glicerolfosfato Deshidrogenasa/genética , OperónRESUMEN
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 (O2â¢-) 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 O2â¢- 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecium/enzimología , Superóxido Dismutasa/metabolismo , Aerobiosis , Animales , Antioxidantes/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Derivados del Benceno/farmacología , Enterococcus faecium/crecimiento & desarrollo , Enterococcus faecium/patogenicidad , Inducción Enzimática , Genoma Bacteriano , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Mariposas Nocturnas/microbiología , Estrés Oxidativo , Filogenia , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Superóxidos/farmacología , VirulenciaRESUMEN
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.
Asunto(s)
Enterococcus faecalis/metabolismo , Glicerol/metabolismo , Aerobiosis , Anaerobiosis , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Glicerolfosfato Deshidrogenasa/genética , Glicerolfosfato Deshidrogenasa/fisiología , Peróxido de Hidrógeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transducción de Señal/genética , Transducción de Señal/fisiología , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/fisiologíaRESUMEN
Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 degrees C versus 37 degrees C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.
Asunto(s)
Infecciones por Actinomycetales/veterinaria , Proteínas Bacterianas/genética , Enfermedades de los Caballos/microbiología , Proteoma , Rhodococcus equi/genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Enzimas/química , Caballos , Immunoblotting , Espectrometría de Masas , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Rhodococcus equi/crecimiento & desarrollo , Rhodococcus equi/inmunología , Homología de Secuencia de Aminoácido , Temperatura , TripsinaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/química , Enterococcus faecalis/química , Proteínas de la Membrana/química , ProteómicaRESUMEN
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.
Asunto(s)
Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/metabolismo , Especies Reactivas de Oxígeno/metabolismo , beta-Lactamas/farmacología , Amoxicilina/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Estrés Oxidativo/efectos de los fármacos , Vitamina K 2/análogos & derivados , Vitamina K 2/farmacologíaRESUMEN
Ers has been qualified as the PrfA-like transcriptional regulator of Enterococcus faecalis. In a previous study we reported that Ers is important for the survival within macrophages of this opportunist pathogenic bacterium. In the present work we have used proteomic and microarray expression profiling of E. faecalis JH2-2 and an ers-deleted mutant (Delta ers mutant) strains to define the Ers regulon. In addition to EF_0082 (encoding a putative facilitator family transporter), already known to be under Ers regulation, three genes or operons displayed a significant decrease (confirmed by reverse transcription quantitative PCR) in expression in the Delta ers mutant. The first locus corresponds to three genes: arcA, arcB, and arcC1 (arcABC). These genes are members of the ADI operon, encoding enzymes of the arginine deiminase system. The second is the EF_1459 gene, which encodes a hypothetical protein and is located within a putative phage genetic element. Lastly, Ef_3319 is annotated as the alpha subunit of the citrate lyase encoded by citF. citF is a member of a putative 12-gene operon involved in citrate catabolism. Moreover, the promoter sequence, similar to the "PrfA box" and found in the promoter regions of ers and EF_0082, has been shown to be included in the DNA segment recognized by Ers. Phenotypic analysis of the Delta ers mutant strain revealed a growth defect when cultured with arginine or citrate as the energy source; this was not seen for the wild type. As expected, similar results were obtained with mutants in which arcA and citF were inactivated. In addition, in the mouse peritonitis model of virulence, the Delta ers mutant appeared significantly less lethal than the JH2-2 wild-type strain. Taken together, these results indicate that the regulator Ers has a pleiotropic effect, especially in the cellular metabolism and virulence of E. faecalis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/metabolismo , Regulación Bacteriana de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Terminación de Péptidos/metabolismo , Proteómica , Regulón , Arginina/metabolismo , Proteínas Bacterianas/genética , Citratos/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Perfilación de la Expresión Génica , Humanos , Mutación , Factores de Terminación de Péptidos/genética , VirulenciaRESUMEN
Cyclopropane fatty acid synthase (cfa) catalyses the transfer of a methyl group from S-adenosylmethionine (SAM) to unsaturated fatty acids. Northern blot experiments demonstrated that the Lactococcus lactis MG1363 cfa gene is mainly expressed as a bicistronic transcript together with metK, the gene encoding SAM synthetase, and is highly induced by acidity. The cfa promoter was characterized by 5'-RACE PCR, and fused to beta-galactosidase by cloning into the pAK80 plasmid. This transcriptional fusion was highly induced by acidity (23-fold at pH 5) as well as during entry into the stationary phase (8-fold) in L. lactis. Interestingly, the cfa promoter expression is repressed in a L. lactis relA* mutant which accumulates (p)ppGpp, whereas its induction by acidity appeared independent of (p)ppGpp in L. lactis and in Escherichia coli.
Asunto(s)
Lactococcus lactis/genética , Metiltransferasas/genética , Adaptación Fisiológica , Secuencia de Bases , ADN Bacteriano , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Concentración de Iones de Hidrógeno , Lactococcus lactis/enzimología , Ligasas/genética , Metionina Adenosiltransferasa/genética , Datos de Secuencia Molecular , Operón , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión , Transcripción Genética , Regulación hacia Arriba , beta-Galactosidasa/genéticaRESUMEN
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.