Discovery of alcohol dehydrogenase (ADH) as a potential vaccine target from Mycolicibacterium smegmatis extracellular vesicles via immunoproteomics.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, releases extracellular vesicles (EVs) that impair macrophage functions and circulate bacterial components to modulate the host immune response. While EVs are increasingly investigated as new vaccines and biomarkers, studying MTB EVs is challenging due to the slow growth rate and pathogenic properties of MTB. Mycolicibacterium smegmatis (MSMEG), a non-pathogenic surrogate with a faster growth rate, offers a safer and more convenient option for laboratory studies due to its similarities to MTB. In this study, we explore the antigenic properties of MSMEG EVs to assess their potential use in developing safer tuberculosis vaccine strategies. Through an immunoproteomics approach that combines comprehensive protein separation by OFFGELTM fractionation, Western blot analysis and mass spectrometry, we identified alcohol dehydrogenase (ADH) - a 46 kDa protein involved in mycobacterial cell wall synthesis - as an antigenic protein from MSMEG EVs. Our findings suggest that MSMEG EVs-derived ADH could improve tuberculosis vaccine formulations and potentially be used for coimmunization with the BCG vaccine, offering new and safer strategies to combat tuberculosis.