Acetyl-Proteomic Characterization of MRSA in Interaction with C. butyricum Unveils ThrS Acetylation Enhancing Bacterial Stress Resistance

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) poses a growing clinical threat because of multidrug resistance. This study investigated the molecular basis of MRSA inhibition by the probiotic Clostridium butyricum (C. butyricum, CB) using quantitative proteomic and acetyl-proteomic analyses, combined with functional characterization of acetylation-mimicking mutations. Proteomic profiling revealed that coculture with C. butyricum substantially altered MRSA protein expression in pathways associated with oxidative phosphorylation, ribosome function, and arginine biosynthesis. Acetyl-proteomic analysis identified the acetylation of ThrS and ClpL upon microbial interaction. To assess the functional impact of these modifications, we generated acetylation-mimicking mutants at specific lysine residues (K18Q for ThrS and K592Q for ClpL), both of which exhibited reduced enzymatic activity in vitro. Further characterization of the ThrS K18Q mutant demonstrated an increased tolerance to environmental stresses and enhanced colonization in a mouse model. Collectively, these multiomics and functional data suggest that C. butyricum may interfere with MRSA metabolic and translational pathways, while the acetylation-mimetic K18Q mutation of ThrS promotes bacterial fitness under stress conditions, likely through a mechanism independent of the C. butyricum-mediated inhibition. This study provides mechanistic insights into probiotic–pathogen interactions and offers a potential molecular basis for developing probiotic-based strategies against MRSA infections.