clpC-Mediated Translational Control Orchestrates Stress Tolerance and Biofilm Formation in Milk-Originated...

Staphylococcus aureus is an important pathogen that can cause widespread infections as well as severe outbreaks of food poisoning. Recent studies have drawn attention to foodborne pathogens such as S. aureus endowed with the ability to form biofilms and increase resistance to antimicrobial agents as well as environmental stress, posing challenges to food safety. The Clp (caseinolytic protease) protein complex plays a crucial role in energy-dependent protein hydrolysis processes. This mechanism is a common way to maintain intracellular homeostasis and regulation in both prokaryotic and eukaryotic cells, especially under stress conditions. In S. aureus, multiple genes encoding Clp ATPase homologues have been identified: clpC, clpB, clpY, clpX, and clpL. This study investigated the roles of clpC in stress tolerance and biofilm formation of foodborne S. aureus RMSA24 isolated from raw milk. Our results showed that the deletion of the clpC gene significantly reduced the bacterium's tolerance to heat, desiccation, hydrogen peroxide, and high osmotic pressure compared to wild type (WT). Furthermore, the clpC knockout mutant also exhibited a marked decrease in biofilm formation using Crystal Violet Staining (CVS) and Scanning Electron Microscopy (SEM). Finally, compared to WT, there was a total of 102 DEGs (differentially expressed genes), with a significant downregulation of genes related to biofilm formation (isaA and spa) and heat-shock response (clpP and danJ). These findings suggest that clpC regulates environmental tolerance in S. aureus by modulating the expression of stress- and biofilm-related genes, positioning it as a potential biomarker and a novel target for controlling contamination in the food industry.