ABSTRACT: Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Persisters are phenotypic variants of bacteria that are able to endure high concentrations of antibiotics, to which they are nonetheless fully susceptible. Most antibiotics require active targets in order to eradicate bacteria. As such, a minimized energy consumption leads to metabolic quiescence and can increase tolerance to antibiotics. Phosphotransfer is a common mechanism to regulate cellular functions, including the most energy intensive process - protein synthesis. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) fine-tune the downregulation of translation during sporulation of Bacillus subtilis, an extreme form of quiescence. Here, we show that deletion of the only annotated functional eukaryotic-like STP, Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity after different stress challenges including acidic pH, intracellular milieu and murine abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphoprotein enrichment and mass spectrometry, we were able to identify possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins and the essential translation elongation factor EF-G. Finally, we show that acid stress exposure leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlated to increased antibiotic persistence. Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Persisters are phenotypic variants of bacteria that are able to endure high concentrations of antibiotics, to which they are nonetheless fully susceptible. Most antibiotics require active targets in order to eradicate bacteria. As such, a minimized energy consumption leads to metabolic quiescence and can increase tolerance to antibiotics. Phosphotransfer is a common mechanism to regulate cellular functions, including the most energy intensive process - protein synthesis. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) fine-tune the downregulation of translation during sporulation of Bacillus subtilis, an extreme form of quiescence. Here, we show that deletion of the only annotated functional eukaryotic-like STP, Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity after different stress challenges including acidic pH, intracellular milieu and murine abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphoprotein enrichment and mass spectrometry, we were able to identify possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins and the essential translation elongation factor EF-G. Finally, we show that acid stress exposure leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlated to increased antibiotic persistence.
Markus Huemer