Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages. On macrophages and T cells, Mtb lipoproteins and lipoglycans regulate many co-stimulatory and inhibitory effects, but the mechanisms for their release and trafficking from infected cells during infection are poorly understood. Here, we demonstrated that Mtb infected macrophages release two distinct populations of extracellular vesicles. One population contains host cell content, while the other contains Mtb molecules which we determine are derived by bacterial vesicle budding. Release of bacterial vesicles is an active process dependent on bacteria viability, not a by-product of Mtb degradation within phagosomes. Inhibition or loss of the host proteins CD14 or CD36 or the Mtb lipoprotein LprG does not affect release of Mtb content from infected macrophages. We propose that bacterial vesicles are secreted by Mtb within infected macrophages and subsequently released into the extracellular environment.
Suppression of CD4+ T cell responses by Mtb contributes to immune evasion. Mtb cell envelope lipoglycans inhibit T cell receptor signaling. We show that bacterial vesicles released from Mtb infected macrophages inhibited activation of CD4+ T cells, demonstrating a mechanism for trafficking of inhibitory lipoglycans to T cells. Utilizing axenic derived bacterial vesicles, we show that lipoglycans from Mtb bacterial vesicles are transferred to T cells membranes. Primary stimulation of naive CD4+ T cells in the presence of BVs induced a state of T cell anergy. We show that Th1 cells, vital for limiting Mtb infection, are inhibited in their activation by bacterial vesicles, but do not have lasting impairment to restimulation.
These studies suggest that bacterial vesicles are the primary mechanism for the export of lipoglycans and lipoproteins from Mtb. These vesicles serve to impair effector functions within infected macrophages, and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells. Further, we demonstrate a novel mechanism for the direct regulation of CD4+ T cells by Mtb lipoglycans conveyed by bacterial vesicles that are produced by Mtb and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.
Committee: Clifford Harding, MD/PhD (Advisor); George Dubyak, PhD (Committee Chair); W Henry Boom, MD (Committee Member); Roxana Rojas, MD/PhD (Committee Member); Pamela Wearsch, PhD (Committee Member); Liem Nguyen, PhD (Committee Member); Clive Hamlin, PhD (Committee Member)