Analysis of cell wall architecture and metabolism of a vancomycin resistant Staphylococcus aureus strain

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Staphylococcus aureus is a bacterial pathogen that causes a broad range of diseases and that is notorious for its resistance to antibiotic agents. Infections with methicillin-resistant S. aureus strains (MRSA), which are frequently hospital-associated, are often treated with the glycopeptide antibiotics vancomycin or teicoplanin or the lipopeptide antibiotic daptomycin. Unfortunately, MRSA strains are able to acquire mutations that simultanteously confer intermediate resistance to vancomycin (VISA phenotype) and full resistance to daptomycin. It has also been demonstrated that these strains are also better protected against the cationic peptides which constitute the natural antibiotics of the innate immune system (e. g. the antimicrobial peptide LL-37). The project will analyse the cell wall structure of S. aureus VC40 which shows full resistance to both antibiotics (vancomycin MIC: 64 µg/ml and daptomycin MIC: 4 µg/ml). Full genome sequencing of this strain revealed mutations in genes encoding the histidine kinases VraS and WalK. Both proteins are located in the cell membrane, and function as sensor proteins which, in answer to signals that have not been identified so far, control cell envelope related processes. Gene expression profiling indicated an increased expression of the genes that are regulated by these kinases in strain VC40. Reconstitution of the mutation present in the VraS kinase (VraS(VC40)) into the susceptible S. aureus NCTC 8325 background resulted in a considerably increased resistance to vancomycin, teicoplanin and daptomycin with MICs surpassing the breakpoints for these antibiotics, thereby generating a VISA (vancomycin intermediary resistant S. aureus) strain. S. aureus VC40 as well as the vraS mutant of strain NCTC 8325 are characterized by a thickened cell wall and a reduced autolytic activity. In order to describe this resistance mechanism further and obtain insight into the influence of the VraSR and WalRK system on cell wall structure, the project will analyse the composition of the bacterial cell wall polymers peptidoglycan, teichoic acid and lipoteichoic acid in strain VC40 and its descendants, clinical isolates, as well as strains that overexpress VraSR and WalRK. Furthermore, the activity of the autolytic enzymes that control the turn-over of the murein and their role in resistance will be investigated as well as the membrane and extracellular protein composition of the strains. The project will attempt to present a comprehensive model of VISA cell wall architecture and metabolism as mediated by VraSR and WalRK.