Disulfide bonds are required for cell division, cell envelope biogenesis and antibiotic resistance proteins in mycobacteria.

Mycobacteria, including Mycobacterium tuberculosis-the etiological agent of tuberculosis-have a unique cell envelope critical for their survival and resistance. The cell envelope's assembly and maintenance influence permeability, making it a key target against multidrug-resistant strains. Disulfide bond (DSB) formation is crucial for the folding of cell envelope proteins. The DSB pathway in mycobacteria includes two enzymes, DsbA and VKOR, required for survival. Using bioinformatics and cysteine profiling proteomics, we identified cell envelope proteins dependent on DSBs. We validated via in vivo alkylation that key proteins like LamA (MmpS3), PstP, LpqW, and EmbB rely on DSBs for stability. Furthermore, chemical inhibition of VKOR results in phenotypes similar to those of Δvkor. Thus, targeting DsbA-VKOR systems could compromise both cell division and mycomembrane integrity. These findings emphasize the potential of DSB inhibition as a novel strategy to combat mycobacterial infections.