Summary Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB) in humans, releases extracellular vesicles in vitro and in vivo. Like extracellular vesicles released by Gram-positive bacteria mycobacterial extracellular vesicles originate at the plasma membrane and are therefore, refer to as membrane vesicles (MV). Mycobacterial MVs contain a broad range of immunologically active proteins and glycolipids. When added to cells in culture, isolated MVs can regulate the immune response of uninfected macrophages, T-cells and dendritic cells. However, the influence of MVs on the interaction(s) between Mtb and the infected macrophage and the overall relevance of MVs production during infection is unclear, mainly because MV-deficient Mtb mutants have not yet been described. It has been recognized that MV biogenesis in M. tuberculosis is an active and regulated process, but the molecular mechanisms and factors involved remain largely unknown. Our previous work demonstrated that iron limitation, a condition encountered in the host, induces the production of MVs in Mtb. Preliminary studies based on this finding identified a pair of iron-responsive, mycobacterial dynamin-like protein (DLP)-encoding genes, isoniazid‐inducible gene A and C (iniAC) that are necessary for MV production. Our preliminary data indicate that a DLP null mutant grows normally in low-iron conditions and in macrophages. However, it exhibits a substantial defect in MV biogenesis. We postulate that this mutant will help define the role of MVs in immunomodulation and pathogenic interactions with the host. The proposed research will test our central hypothesis that MV production during macrophage infection requires the DLPs IniAC and that comparison of the immune response associated with macrophages infected with WT Mtb or the MV-deficient iniAC mutant will reveal substantial differences. The outcome of this project will increase understanding of MV biogenesis in Mtb and its relevance during macrophage infection, and provide the foundation for in vivo studies that evaluate the relevance of MVs at the Mtb-host interface.