Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureus infections. Several transcriptomics and proteomics studies have investigated the bacterium's cellular response to vancomycin, but quantitative proteomic studies have been limited in the number of proteins and restricted to certain sub-cellular compartments so far.

Here, we combined the enrichment of different proteomic sub-fractions with in vivo metabolic labeling and shotgun proteomics to analyze the vancomycin induced stress response. Quantitative data for approximately 1400 proteins could be obtained, covering the majority of cytosolic as well as membrane localized proteins, cell surface associated and extracellular proteins. Besides major adaptive processes induced by limited growth of the cells due to the sublethal vancomycin exposure, specific cellular responses are seen on proteome level, e.g. the specific increase of proteins synthesizing amino acids which are essential for the peptidoglycan synthesis or the decrease of most proteins with a virulence related function. Most important, the influence on regulatory targets of the two-component system VraSR as the main regulatory system known for cell wall stress as well as for global regulons like SigB and SaeR was detected.