Systematic analysis of CHAP domain-containing genes deletion mutants links cell wall architecture to antibiotic susceptibility and pathogenesis in S. aureus.

Staphylococcus aureus is a major human pathogen whose resilience is largely attributed to a robust and dynamically remodeled cell wall. CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain-containing proteins constitute a large family of peptidoglycan hydrolases implicated in cell wall turnover, but their precise physiological roles remain poorly defined due to functional redundancy and limitations of prior mutagenesis approaches. Here, we performed a systematic functional analysis of CHAP proteins in the epidemic methicillin-resistant S. aureus USA300 JE2 by constructing clean, markerless deletions of 13 CHAP-encoding genes and selected double mutants. Phenotypic profiling revealed that CHAP proteins are not essential for growth but significantly influence β-lactam susceptibility, autolysis, biofilm formation, and virulence. Notably, deletion of sle1 (SAUSA300_0438) and SsaA-family genes (SAUSA300_2249, SAUSA300_2503) led to distinct alterations in penicillin-binding protein (PBP) expression, increased β-lactam sensitivity, and impaired biofilm development. Transmission electron microscopy demonstrated that these mutants exhibited significant changes in cell wall thickness, cell size, and septation dynamics, linking structural perturbations to observed functional defects. In a Galleria mellonella infection model, several CHAP mutants showed attenuated virulence. Collectively, our study establishes CHAP hydrolases as specialized regulators of cell envelope homeostasis and virulence in S. aureus, providing a foundation for their exploitation as targets for novel anti-staphylococcal strategies.