RESUMO
Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Brônquios/citologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , Proteômica/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/isolamento & purificação , Linhagem Celular , Humanos , Proteoma/isolamento & purificação , Proteômica/economia , Fatores de TempoRESUMO
The species Staphylococcus aureus harbors 19 superantigen gene loci, six of which are located in the enterotoxin gene cluster (egc). Although these egc superantigens are far more prevalent in clinical S. aureus isolates than non-egc superantigens, they are not a prominent cause of toxic shock. Moreover, neutralizing Abs against egc superantigens are very rare, even among carriers of egc-positive S. aureus strains. In search of an explanation, we have tested two non-exclusive hypotheses: 1) egc and non-egc superantigens have unique intrinsic properties and drive the immune system into different directions and 2) egc and non-egc superantigens are released by S. aureus under different conditions, which shape the immune response. A comparison of three egc (SEI, SElM, and SElO) and three non-egc superantigens (SEB, SElQ, and toxic shock syndrome toxin-1) revealed that both induced proliferation of human PBMC with comparable potency and elicited similar Th1/Th2-cytokine signatures. This was supported by gene expression analysis of PBMC stimulated with one representative superantigen from each group (SEI and SEB). They induced very similar transcriptional changes, especially of inflammation-associated gene networks, corresponding to a very strong Th1- and Th17-dominated immune response. In contrast, the regulation of superantigen release differed markedly between both superantigen groups. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc superantigens were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc superantigens is not due to their intrinsic properties, which are very similar, but caused by their differential release by S. aureus.