Project Summary - Toxin secretion and trafficking by Mycobacterium tuberculosis The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Most pathogenic bacteria use toxins to evade and/or subvert immune responses, to survive and replicate in the host. CpnT is an outer membrane protein with an N-terminal channel domain and a C-terminal toxin domain (TNT), which causes necrotic cell death in host cells. Up-to-date TNT is the only known exotoxin of Mycobacterium tuberculosis. We showed that TNT is secreted into the cytosol of macrophages infected with M. tuberculosis where it hydrolyzes NAD+. NAD+ depletion by TNT activates necroptosis, a programmed cell death pathway. Importantly, necroptosis-deficient mice are more resistent to infection with M. tuberculosis, underlining the importance of this pathway for tuberculosis pathogenesis. Permeabilization of the phagosomal membrane is a critical step in the pathogenesis of M. tuberculosis, not only because it required for M. tuberculosis proteins such as the toxin TNT to reach the cytosol of infected cells, but it also enables M. tuberculosis to escape from the phagosome resulting in bacterial dissemination. Thus, TNT secretion and trafficking are an important components of the intracellular survival strategy of M. tuberculosis and for tuberculosis pathogenesis. This research project will reveal novel molecular mechanisms of toxin secretion and trafficking by M. tuberculosis and the role of these mechanisms in virulence and pathogenesis of M. tuberculosis.