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

Abstract

ABSTRACTMycobacterium tuberculosis (Mtb) 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 Mtb by analyzing Mtb 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 iniBAC operon in association with high vesicle production in Mtb 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 Mtb 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 Mtb-derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research.IMPORTANCEIron is an essential nutrient that promotes survival and growth of M. tuberculosis, the bacterium that causes human tuberculosis (TB). Limited availability of iron, often encountered in the host environment, stimulates M. tuberculosis to secrete membrane-bound extracellular vesicles containing molecules that may help it evade the immune system. Characterizing the bacterial factors and mechanisms involved in the production of mycobacterial vesicles is important for envisioning ways to interfere with this process. Here, we report the discovery of proteins required by M. tuberculosis for vesicle biogenesis in culture and during host cell infection. We also demonstrate a connection between antibiotic response and extracellular vesicle production. The work provides insights into the mechanisms underlying vesicle biogenesis in M. tuberculosis and permits better understanding of the significance of vesicle production to M. tuberculosis-host interactions and antibiotic stress response.