Cholesterol-activated VapC12 toxin modulates growth and drug susceptibility in Mycobacterium tuberculosis

Tuberculosis eradication efforts are severely hampered by antimicrobial resistance and bacterial persistence. We previously reported that the VapBC12 toxin antitoxin (TA) system in Mycobacterium tuberculosis (M. tuberculosis) regulates disease progression and antibiotic persistence. In this study, we investigated the molecular mechanisms underlying VapC12 dependent modulation of bacterial growth and antibiotic susceptibility. We identified a Cholesterol Recognition Amino acid Consensus (CRAC) motif in the VapC12 toxin that is critical for cholesterol binding and interaction with its cognate antitoxin, VapB12. Heterologous expression of VapC12 in Mycobacterium smegmatis induced toxicity, providing a model to investigate its functional activity. We determined the crystal structure of VapB12 at 1.6 Angstorm resolution and used molecular modelling to predict the VapBC12 complex structure. Structure-function analyses identified specific residues critical for complex assembly and toxin activity. Mechanistically, our findings suggest that cholesterol activates VapC12 by competitively displacing VapB12 from the CRAC motif, thereby restricting M. tuberculosis growth. Finally, we showed that pharmacologically inhibiting VapC12 alleviates cholesterol-mediated growth restriction and enhances antibiotic activity. Collectively, these findings establish a mechanistic link between cholesterol sensing, toxin-antitoxin regulation, and antibiotic susceptibility in M. tuberculosis, highlighting VapC12 as a potential target for therapeutic strategies to combat tuberculosis persistence.