Using BONCAT To Dissect The Proteome OfS. aureusPersisters

Abstract

Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. As such, it is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation, yet identifying the proteins expressed during the induction of the persister state has been difficult, because the persister-state is rare, transient and does not lead to genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label, and retrieve, the proteome expressed during the persister state for different strains of methicillin-resistant Staphylococcus aureus. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome fraction of Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) under challenge with β-lactam and fluoroquinolone antibiotics with Label Free Quantification - Liquid chromatography mass spectrometry (LFQ-LCMSased proteomics reveals the upregulation of proteins involved in stringent response, cell wall biosynthesis, purine metabolism, ppGpp biosynthesis, two component systems (TCS), lipid metabolism, ABC transporters, d-alanine biosynthesis and L-proline degradation. Conversely, we observed a decline of proteins associated with amino acid biosynthesis and degradation, protein biosynthesis, protein modification, and carbohydrate metabolism, among others. These findings indicate that modification of translational activity in persister cells enables bacterial cells to induce an active defense to survive antibiotic pressure.