| One third of the world's population is infected with Mycobacterium tuberculosis, causing two million deaths annually. The bacteria avoid immune clearance by persisting within macrophages by subverting normal phagosome maturation and acidification. In order to spread, the bacteria induce necrotic death of its host macrophage, broadcasting the infection into neighboring cells. However, it has long been appreciated that the apoptotic, rather than necrotic death of an infected macrophage results in bacterial growth suppression, improved adaptive immune response and survival. The mechanism for apoptosis-mediated bacterial suppression has hitherto remained unknown.;In this dissertation we report that apoptosis itself is not intrinsically bactericidal. We find that following apoptosis, the M. tuberculosis -infected macrophage is engulfed by bystander macrophages through the process of efferocytosis. Efferocytosis, or apoptotic cell clearance, is a critical function of macrophages; however, little is known regarding efferocytosis of infected apoptotic cells. We find that M. tuberculosis -infected macrophages die by apoptosis more commonly than found previously. By confocal microscopy we observed that apoptotic macrophages are rapidly engulfed by uninfected macrophages. Efferocytosis of M. tuberculosis -infected macrophages occurs in vitro with all macrophage types tested and in vivo- specifically in the lung, indicating that efferocytosis could play an important role during infection. We developed an uninfected macrophage co-culture system in which we observe efferocytosis and define conditions in which it occurs. Using this co-culture system we observe a suppression of bacterial growth. Blocking efferocytosis using three different strategies, we have found that the engulfment of infected cells is required for M. tuberculosis control in the macrophage co-culture system, demonstrating that efferocytosis is a novel antibacterial mechanism. We then demonstrated using transmission electron microscopy that the M. tuberculosis-containing efferocytic phagosome is structurally distinct from the traditional M. tuberculosis phagosome. Bacteria from within the efferocytic phagosome are unable to halt its maturation, and as such are delivered to lysosomes with great efficiency. Furthermore, we find that following efferocytosis, M. tuberculosis are killed. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, our work indicates that is should also be viewed as an antimicrobial effector mechanism. |
