Abstract
<jats:p>Endogenous hydrogen sulfide (H2S) serves as a pivotal gasotransmitter conferring antimicrobial resistance and shielding bacteria from oxidative stress. While cystathionine γ-lyase (CSE)-dependent H2S production is critical for Staphylococcus aureus survival, the upstream regulatory nodes linking metabolic status to H2S-mediated redox defense remain elusive. Here, we identify heme A synthase (CtaA) as a master regulator that couples respiratory metabolism with H2S biosynthesis, specifically under glucose-depleted conditions mimicking host niches such as abscesses and phagosomes. Using a Himar1 transposon screen followed by a high efficiency detection method, we demonstrate that CtaA deficiency precipitates a catastrophic collapse in H2S levels (<10% of wild-type), leading to severe virulence and antimicrobial resistance changes. Specifically, the ΔctaA mutant exhibits attenuated hemolytic activity and significantly reduced virulence in a Galleria mellonella model, despite displaying paradoxical resistance to specific antimicrobials. Mechanistically, CtaA deletion triggers a maladaptive metabolic reprogramming characterized by the downregulation of the L-cysteine transporter TcyP and dysregulation of arginine metabolism, which collectively impair the bacterium’s capacity to produce endogenous H2S to defense oxidative stress. Notably, this redox vulnerability is linked to altered endogenous nitric oxide (NO) dynamics, suggesting a disrupted H2S-NO crosstalk essential for stress adaptation. Our findings elucidate a novel metabolic-redox axis where CtaA governs H2S homeostasis to counteract host-imposed oxidative stress. Targeting the CtaA-H2S axis represents a promising therapeutic strategy to sensitize S. aureus to host immune clearance by dismantling its critical redox shield.</jats:p>