Dynamin-like proteins are essential for vesicle biogenesis in Mycobacterium tuberculosis

secretes pathogenicity factors and immunologically active molecules via membrane vesicles. However, nothing is known about the mechanisms involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of membrane vesicle production in by analyzing cells under conditions of high vesicle production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell envelope restructuring in association with vesicle release that correlated with downregulation of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics showed common upregulation of the operon in association with high vesicle production in cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary for release of EV by in culture and in infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis. The findings will enable further understanding of the relevance of -derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research.