Cholesterol-mediated activation of VapC12 toxin modulates growth and drug susceptibility in Mycobacterium tuberculosis

Tuberculosis eradication efforts are severely hampered by antimicrobial resistance (AMR). We have previously reported that VapBC12 TA system in Mycobacterium tuberculosis (M. tuberculosis) regulates both disease and antibiotic persistence in tuberculosis. In this study, we developed a mechanistic understanding of the VapBC12-dependent modulation of growth and antibiotic susceptibility in M. tuberculosis. We identified a unique Cholesterol Recognition Amino acid Consensus (CRAC) motif in VapC12 toxin and demonstrated that its heterologous expression induces toxicity in M. smegmatis. We have solved the crystal structure of VapB12 antitoxin at a 1.6A0 wavelength and using molecular modeling predicted the structure of TA complex. Structure-function analysis revealed specific residues critical for the assembly and activity of the VapBC12 TA system. Our study suggests that cholesterol activates the VapC12 toxin by competitively displacing its cognate antitoxin at the CRAC motif which consequently restricts the growth of M. tuberculosis. Finally, we demonstrated that chemical inhibition of VapC12 prevents growth restriction and potentiates activity of anti-TB drugs.