RESUMO
Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.
Assuntos
Aderência Bacteriana/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/metabolismo , Mutação/genética , Proteômica , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genótipo , Ferro/farmacologia , Cinética , Lactococcus/efeitos dos fármacos , Lactococcus/metabolismo , Proteínas de Membrana/genética , Viabilidade Microbiana/efeitos dos fármacos , Biblioteca de Peptídeos , Peptídeos/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Tripsina/metabolismoRESUMO
Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1ß, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1ß and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.
Assuntos
Adesinas Bacterianas/metabolismo , Bacteriemia/imunologia , Endocardite Bacteriana/imunologia , Agregação Plaquetária/imunologia , Staphylococcus aureus/metabolismo , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana/fisiologia , Citocinas/genética , Citocinas/metabolismo , Fibrinogênio , Fibronectinas , Regulação da Expressão Gênica/imunologia , Humanos , Proteínas Imobilizadas , RatosRESUMO
Levels of the enzymes that produce wound response mediators have to be controlled tightly in unwounded tissues. The Arabidopsis (Arabidopsis thaliana) fatty acid oxygenation up-regulated8 (fou8) mutant catalyzes high rates of alpha -linolenic acid oxygenation and has higher than wild-type levels of the alpha -linolenic acid-derived wound response mediator jasmonic acid (JA) in undamaged leaves. fou8 produces a null allele in the gene SAL1 (also known as FIERY1 or FRY1). Overexpression of the wild-type gene product had the opposite effect of the null allele, suggesting a regulatory role of SAL1 acting in JA synthesis. The biochemical phenotypes in fou8 were complemented when the yeast (Saccharomyces cerevisiae) sulfur metabolism 3'(2'), 5'-bisphosphate nucleotidase MET22 was targeted to chloroplasts in fou8. The data are consistent with a role of SAL1 in the chloroplast-localized dephosphorylation of 3'-phospho-5'-adenosine phosphosulfate to 5'-adenosine phosphosulfate or in a closely related reaction (e.g. 3',5'-bisphosphate dephosphorylation). Furthermore, the fou8 phenotype was genetically suppressed in a triple mutant (fou8 apk1 apk2) affecting chloroplastic 3'-phospho-5'-adenosine phosphosulfate synthesis. These results show that a nucleotide component of the sulfur futile cycle regulates early steps of JA production and basal JA levels.
Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Folhas de Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Mutação , Monoéster Fosfórico Hidrolases/genética , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
A statistical methodology for the objective comparison of LDI-MS mass spectra of blue gel pen inks was evaluated. Thirty-three blue gel pen inks previously studied by RAMAN were analyzed directly on the paper using both positive and negative mode. The obtained mass spectra were first compared using relative areas of selected peaks using the Pearson correlation coefficient and the Euclidean distance. Intra-variability among results from one ink and inter-variability between results from different inks were compared in order to choose a differentiation threshold minimizing the rate of false negative (i.e. avoiding false differentiation of the inks). This yielded a discriminating power of up to 77% for analysis made in the negative mode. The whole mass spectra were then compared using the same methodology, allowing for a better DP in the negative mode of 92% using the Pearson correlation on standardized data. The positive mode results generally yielded a lower differential power (DP) than the negative mode due to a higher intra-variability compared to the inter-variability in the mass spectra of the ink samples.
RESUMO
L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.
Assuntos
Aminobutiratos/metabolismo , Antibacterianos/biossíntese , Antimetabólitos/metabolismo , Vias Biossintéticas/genética , Família Multigênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ordem dos Genes , Genes Bacterianos , Mutagênese Sítio-DirigidaRESUMO
Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q(554)E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.
Assuntos
Mutação , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Penicilinas/farmacologia , Streptococcus/efeitos dos fármacos , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Humanos , Mastite Bovina/microbiologia , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/genéticaRESUMO
INTRODUCTION: Cefepime has been associated with a greater risk of mortality than other beta-lactams in patients treated for severe sepsis. Hypotheses for this failure include possible hidden side-effects (for example, neurological) or inappropriate pharmacokinetic/pharmacodynamic (PK/PD) parameters for bacteria with cefepime minimal inhibitory concentrations (MIC) at the highest limits of susceptibility (8 mg/l) or intermediate-resistance (16 mg/l) for pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. We examined these issues in a prospective non-interventional study of 21 consecutive intensive care unit (ICU) adult patients treated with cefepime for nosocomial pneumonia. METHODS: Patients (median age 55.1 years, range 21.8 to 81.2) received intravenous cefepime at 2 g every 12 hours for creatinine clearance (CLCr) >or= 50 ml/min, and 2 g every 24 hours or 36 hours for CLCr < 50 ml/minute. Cefepime plasma concentrations were determined at several time-points before and after drug administration by high-pressure liquid chromatography. PK/PD parameters were computed by standard non-compartmental analysis. RESULTS: Seventeen first-doses and 11 steady states (that is, four to six days after the first dose) were measured. Plasma levels varied greatly between individuals, from two- to three-fold at peak-concentrations to up to 40-fold at trough-concentrations. Nineteen out of 21 (90%) patients had PK/PD parameters comparable to literature values. Twenty-one of 21 (100%) patients had appropriate duration of cefepime concentrations above the MIC (T>MIC >or= 50%) for the pathogens recovered in this study (MIC Assuntos
Antibacterianos/sangue
, Cefalosporinas/sangue
, Unidades de Terapia Intensiva
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Antibacterianos/administração & dosagem
, Antibacterianos/efeitos adversos
, Antibacterianos/farmacocinética
, Antibacterianos/farmacologia
, Cefepima
, Cefalosporinas/administração & dosagem
, Cefalosporinas/efeitos adversos
, Cefalosporinas/farmacocinética
, Cefalosporinas/farmacologia
, Infecção Hospitalar
, Europa (Continente)
, Feminino
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Monitorização Fisiológica/métodos
, Pneumonia/tratamento farmacológico
, Estudos Prospectivos
, Adulto Jovem
RESUMO
BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.
Assuntos
Proteínas de Bactérias/genética , Parede Celular/química , Inativação Gênica , Genes Bacterianos , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Biologia Computacional , Teste de Complementação Genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Polissacarídeos/química , Staphylococcus aureus/metabolismo , Staphylococcus aureus/ultraestrutura , Temperatura , Transcrição GênicaRESUMO
Quinupristin/dalfopristin (Q/D) and ß-lactams interact positively against methicillin-resistant Staphylococcus aureus (MRSA). The effect extends to other inhibitors of protein synthesis, but not to inhibitors of polynucleotide synthesis or assembly, or to Q/D plus non-ß-lactam cell wall inhibitors. Moreover, electron microscopy studies have correlated this effect with a thickened cell wall. In this study, we sought to determine whether inhibitors of protein synthesis might produce a specific peptidoglycan muropeptide signature that would correlate with their positive ß-lactam interaction. The muropeptides of six S. aureus isolates (three methicillin-susceptible and three MRSA) were analysed using high-performance liquid chromatography and mass spectrometry. Exposure to 0.25× the minimum inhibitory concentration of inhibitors of protein synthesis consistently produced three main alterations irrespective of methicillin resistance: (i) an increase in peak 12 (a cyclic dimer of glycine-containing disaccharide-tetrapeptide); (ii) an increase in poorly resolved late-eluting materials; and (iii) a decrease in peak 1 (a disaccharide-pentapeptide). Eventually, the rate of autolysis was also decreased, supporting the structural alteration of the peptidoglycan. Other drug classes did not produce these anomalies. An increase in peak 12 was also observed in staphylococci treated with fosfomycin, which decreases expression of the native penicillin-binding protein (PBP) 2 and 4. Parallel blockage of normal PBPs with ß-lactams abolished the anomalies, indicating that they resulted from altered function of native PBPs. This underlines the potential of inhibiting both protein synthesis and transpeptidation simultaneously and suggests that such a drug combination strategy might be efficaciously exploited.
Assuntos
Antibacterianos/metabolismo , Sinergismo Farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Peptídeos/análise , Peptidoglicano/química , Inibidores da Síntese de Proteínas/metabolismo , Parede Celular/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Testes de Sensibilidade Microbiana , beta-Lactamas/metabolismoRESUMO
To explore the discriminatory power of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detecting subtle differences in isogenic isolates, we tested isogenic strains of Staphylococcus aureus differing in their expression of resistance to methicillin or teicoplanin. More important changes in MALDI-TOF MS spectra were found with strains differing in methicillin than in teicoplanin resistance. In comparison, very minor or no changes were recorded in pulsed-field gel electrophoresis profiles or peptidoglycan muropeptide digest patterns of these strains, respectively. MALDI-TOF MS might be useful to detect subtle strain-specific differences in ionizable components released from bacterial surfaces and not from their peptidoglycan network.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Resistência a Meticilina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus aureus/classificação , Teicoplanina/farmacologia , Técnicas de Tipagem Bacteriana/métodos , Eletroforese em Gel de Campo Pulsado , Humanos , Muramidase/metabolismo , Peptídeos/análise , Peptidoglicano/química , Peptidoglicano/metabolismo , Sensibilidade e Especificidade , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Bacterial programmed cell death and quorum sensing are direct examples of prokaryote group behaviors, wherein cells coordinate their actions to function cooperatively like one organism for the benefit of the whole culture. We demonstrate here that 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a Pseudomonas aeruginosa quorum-sensing-regulated low-molecular-weight excreted molecule, triggers autolysis by self-perturbing the electron transfer reactions of the cytochrome bc1 complex. HQNO induces specific self-poisoning by disrupting the flow of electrons through the respiratory chain at the cytochrome bc1 complex, causing a leak of reducing equivalents to O2 whereby electrons that would normally be passed to cytochrome c are donated directly to O2. The subsequent mass production of reactive oxygen species (ROS) reduces membrane potential and disrupts membrane integrity, causing bacterial cell autolysis and DNA release. DNA subsequently promotes biofilm formation and increases antibiotic tolerance to beta-lactams, suggesting that HQNO-dependent cell autolysis is advantageous to the bacterial populations. These data identify both a new programmed cell death system and a novel role for HQNO as a critical inducer of biofilm formation and antibiotic tolerance. This newly identified pathway suggests intriguing mechanistic similarities with the initial mitochondrial-mediated steps of eukaryotic apoptosis.
Assuntos
Antibacterianos/farmacologia , Biofilmes , Transporte de Elétrons/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidroxiquinolinas/farmacologia , Membranas Mitocondriais/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/fisiologiaRESUMO
High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient > or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.
Assuntos
Antifúngicos/sangue , Fluconazol/sangue , Animais , Celulose , Cromatografia Líquida de Alta Pressão , Humanos , Polímeros , Ratos , Padrões de Referência , Reprodutibilidade dos Testes , Sulfonas , UltrafiltraçãoRESUMO
Pearson correlation coefficients were applied for the objective comparison of 30 black gel pen inks analysed by laser desorption ionization mass spectrometry (LDI-MS). The mass spectra were obtained for ink lines directly on paper using positive and negative ion modes at several laser intensities. This methodology has the advantage of taking into account the reproducibility of the results as well as the variability between spectra of different pens. A differentiation threshold could thus be selected in order to avoid the risk of false differentiation. Combining results from positive and negative mode yielded a discriminating power up to 85%, which was better than the one obtained previously with other optical comparison methodologies. The technique also allowed discriminating between pens from the same brand.
RESUMO
The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.
Assuntos
Parede Celular/metabolismo , Ligases/metabolismo , Staphylococcus aureus/enzimologia , Estresse Fisiológico , Ácidos Teicoicos/metabolismo , Bacitracina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/genética , Meios de Cultura/metabolismo , Deleção de Genes , Fusão Gênica , Genes Bacterianos , Genes Reporter , Teste de Complementação Genética/métodos , Ligases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Regiões Promotoras Genéticas , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tunicamicina/farmacologiaRESUMO
MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.
Assuntos
Parede Celular/fisiologia , Parede Celular/ultraestrutura , Proteínas de Membrana/metabolismo , Staphylococcus aureus/patogenicidade , Propriedades de Superfície , Animais , Antibacterianos/farmacologia , Aderência Bacteriana , Caenorhabditis elegans/microbiologia , Endocardite Bacteriana/microbiologia , Endocardite Bacteriana/patologia , Deleção de Genes , Perfilação da Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , VirulênciaRESUMO
Glycopeptide-intermediate resistant Staphylococcus aureus (GISA) are characterized by multiple changes in the cell wall and an altered expression of global virulence regulators. We investigated whether GISA are affected in their infectivity in a rat model of experimental endocarditis. The glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 and its laboratory-derived GISA M1V16 were examined for their ability to (i) adhere to fibrinogen and fibronectin in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonize aortic vegetations in rats, and (iv) compete for valve colonization by co-inoculation. Both GISA M1V16 and M1V2 adhered similarly to fibrinogen and fibronectin in vitro. In rats, GISA M1V16 was cleared faster from the blood (P < 0.05) and required 100-times more bacteria than parent M1V2 (10(6) versus 10(4)CFU) to infect 90% of vegetations. GISA M1V16 also had 100 to 1000-times lower bacterial densities in vegetations. Moreover, after co-inoculation with GISA M1V16 and M1V2Rif, a rifampin-resistant variant of M1V2 to discriminate them in organ cultures, GISA M1V16 was out-competed by the glycopeptide-susceptible counterpart. Thus, in rats with experimental endocarditis, GISA showed an attenuated virulence, likely due to a faster clearance from the blood and a reduced fitness in cardiac vegetations. The GISA phenotype appeared globally detrimental to infectivity.
Assuntos
Farmacorresistência Bacteriana , Endocardite Bacteriana/microbiologia , Glicopeptídeos/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Antibacterianos/farmacologia , Aderência Bacteriana , Modelos Animais de Doenças , Mutação , Ratos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , VirulênciaRESUMO
The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.
Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Fenóis/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/metabolismo , Sideróforos/biossíntese , Tiazóis/metabolismo , Cisteína/metabolismo , Isomerismo , Estrutura Molecular , Fenóis/síntese química , Fenóis/química , Ácido Salicílico/metabolismo , Sideróforos/síntese química , Sideróforos/química , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.
Assuntos
Arabidopsis/metabolismo , Germinação , Malondialdeído/metabolismo , Meristema/metabolismo , Tocoferóis , Alquil e Aril Transferases/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos , Regulação Enzimológica da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Germinação/genética , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Meristema/genética , Oxirredução , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tiobarbitúricos/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Tocoferóis/metabolismoRESUMO
The canine distemper virus (CDV) belongs to the Morbillivirus genus which includes important human pathogens like the closely related measles virus. CDV infection can reach the nervous system where it causes serious malfunctions. Although this pathology is well described, the molecular events in brain infection are still poorly understood. Here we studied infection in vitro by CDV using a model of dissociated cell cultures from newborn rat hippocampus. We used a recombinant CDV closely related to the neurovirulent A75/17 which also expresses the enhanced green fluorescent protein. We found that infected neurons and astrocytes could be clearly detected, and that infection spreads only slowly to neighboring cells. Interestingly, this infection causes a massive cell death of neurons, which includes also non-infected neurons. Antagonists of NMDA-type or alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA)-type glutamate receptors could slow down this neuron loss, indicating an involvement of the glutamatergic system in the induction of cell death in infected and non-infected cells. Finally, we show that, following CDV infection, there is a steady increase in extracellular glutamate in infected cultures. These results indicate that CDV infection induces excitotoxic insults on neurons via glutamatergic signaling.
Assuntos
Cinomose/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/virologia , Degeneração Neural/metabolismo , Degeneração Neural/virologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/virologia , Morte Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Cinomose/fisiopatologia , Vírus da Cinomose Canina/patogenicidade , Vírus da Cinomose Canina/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Líquido Extracelular/metabolismo , Gliose/metabolismo , Gliose/fisiopatologia , Gliose/virologia , Proteínas de Fluorescência Verde , Hipocampo/fisiopatologia , Degeneração Neural/fisiopatologia , Neurônios/metabolismo , Neurônios/virologia , Ratos , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Proteínas Recombinantes , Regulação para Cima/fisiologia , Células VeroRESUMO
Streptococcus gordonii alpha-phosphoglucomutase, which converts glucose 6-phosphate to glucose 1-phosphate, is encoded by pgm. The pgm transcript is monocistronic and is initiated from a sigma(A)-like promoter. Mutants with a gene disruption in pgm exhibited an altered cell wall muropeptide pattern and a lower teichoic acid content, and had reduced fitness both in vitro and in vivo. In vitro, the reduced fitness included reduced growth, reduced viability in the stationary phase and increased autolytic activity. In vivo, the pgm-deficient strain had a lower virulence in a rat model of experimental endocarditis.