Staphylococcus aureus is the leading cause of infections worldwide and infection results in a variety of diseases. As of no surprise, phosphorylation is a major game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-like serine/threonine kinases have been implicated in these complex signaling cascades. Due to the sub-stoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is, however, to date still limited.
Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe3+-IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome dataset of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites we were able to increase the number of identifications by more than 21 times compared to recent literature. In addition, we were able to identify downstream targets of the only known Ser/Thr kinase of the S. aureus strain USA300, Stk1. Together this work allowed an extensive analysis of the bacterial phosphoproteome and will help to understand mechanisms involved in virulence and antibiotic resistance.