RESUMO
Legionella pneumophila, the agent of Legionnaires' disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.
Assuntos
Dictyostelium/química , Dictyostelium/microbiologia , Legionella/crescimento & desenvolvimento , Fagossomos/química , Fagossomos/microbiologia , Proteoma/análise , Proteínas de Protozoários/análise , Actinas/metabolismo , Animais , Fracionamento Celular , Inibidores de Cisteína Proteinase/metabolismo , Dictyostelium/fisiologia , Dictyostelium/ultraestrutura , Eletroforese em Gel Bidimensional , Genômica/métodos , Lisossomos/enzimologia , Lisossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Biológicos , NADPH Oxidases/farmacologia , Fagossomos/diagnóstico por imagem , Fagossomos/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteoma/isolamento & purificação , Proteômica/métodos , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxidos/metabolismo , UltrassonografiaRESUMO
Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase-mediated effect on post-stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488-4496). In the present study, the global function of ClpC in physiology, metabolism, and late-stationary phase survival was examined using DNA microarrays and 2-D PAGE followed by MALDI-TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose-phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell-associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late-stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.
Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/fisiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Carbono/metabolismo , Carbono/fisiologia , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/fisiologia , Fenótipo , Staphylococcus aureus/genética , Fatores de TempoRESUMO
Staphylococcus epidermidis is a commensal bacterium of the human skin. However, S. epidermidis and other coagulase-negative staphylococci (CNS) emerge also as common nosocomial pathogens infecting immunocompromized patients carrying medical devices. Antibiotic resistance and the ability of many nosocomial S. epidermidis isolates to form biofilms on inert surfaces make these infections hard to treat. Epidemiological analyses using multilocus sequence typing (MLST) and genetic studies suggest that S. epidermidis isolates in the hospital environment differ from those obtained outside of medical facilities with respect to biofilm formation, antibiotic resistance, and the presence of mobile DNA elements. Since S. epidermidis isolates exhibit high genome flexibility, they are now regarded as reservoirs for the evolution and spread of resistance traits within nosocomial bacterial communities.
Assuntos
Infecção Hospitalar/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/patogenicidade , Biofilmes , Infecção Hospitalar/tratamento farmacológico , Variação Genética , Humanos , Resistência a Meticilina/genética , Epidemiologia Molecular , Fenótipo , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genéticaRESUMO
The quorum-sensing system Agr is part of a complex regulatory network of gene expression in staphylococci. This study presents the effect of an agr mutation on a biofilm-forming Staphylococcus epidermidis isolate by employing proteome and transcriptome analysis. The agr mutant exhibited a significantly lowered amount of extracellular proteins: amongst others SspA, AtlE, GehD and the phenol soluble modulins PSM1/2. Cytoplasmic proteome analysis and expression profiling indicated that the agr inactivation led to a strongly altered regulation of metabolism and virulence. Most strikingly, expression of CodY, a global regulator of virulence and stationary phase gene expression, was decreased in the agr mutant. In this respect, homologous genes known to be controlled by CodY in Bacillus subtilis and Lactococcus lactis were found to be up-regulated in the S. epidermidis agr mutant. The combined data show that wild-type and agr mutant differ with respect to amino acid biosynthesis and oligopeptide transport, carbohydrate utilization, as well as GMP and IMP interconversion. Due to the varying physiological properties S. epidermidis agr mutants, which often occur spontaneously, might be capable of colonizing alternative ecological niches in the human host and could, therefore, have an advantage in adapting to changing environmental conditions.