Formation of CO2-dependent small-colony variants (SCVs) in methicillin-resistant Staphylococcus aureus mediated by a single point mutation in mpsB

Staphylococcus aureus is a major human pathogen and the emergence of small colony variants (SCVs) makes the treatment of S. aureus infections more challenging. The mechanism of CO 2 -dependent SCVs (CO 2 -SCVs) remains inadequately explored. In this study, a clinical CO 2 -SCV strain (GY8) and a normal strain (GY9) were characterized from a patient with long-term infection. Through whole-genome sequencing, we detected a point mutation (H607Y) in the mpsB gene associated with the SCV phenotype. Transcriptomic profiling of both strains was performed to identify key genes or pathways closely related to SCV formation, followed by functional validation. The mpsB (H607Y) mutant exhibited typical SCV characteristics, including reduced growth rate, decreased pigmentation, and increased aminoglycoside resistance. Compared to GY9, the SCV strain GY8 showed attenuated virulence but enhanced intracellular persistence, facilitating chronic infection. Additionally, the mutant displayed heightened sensitivity to Triton X-100 induced cell lysis, though electron microscopy revealed significant changes in cell wall thickness..under ambient air. Finally, protein structure prediction and molecular docking analysis revealed that the H607 residue likely serves as the active center of MpsB, providing new mechanistic insights into the formation of CO 2 -SCVs. Our findings demonstrate that a single mpsB mutation drives SCV emergence, highlighting its role in persistent infections and providing potential targets for therapeutic intervention.