Tumor necrosis factor (TNF) is a key host factor in tuberculosis (TB). In the zebrafish model for TB, excess TNF triggers necrosis of infected macrophages which leads to exuberant extracellular bacterial growth and host susceptibility. In infected macrophages, excess TNF induces production of reactive oxygen species (ROS) which activate two death pathways one of which involves the mitochondrial transition pore, and the other leads to ceramide production and lysosomal permeabilization. In exploring the events downstream of ceramide production we found that the pro-apoptotic factor Bax is required for macrophage necrosis in excess TNF conditions. Bax has been well-characterized for its role during apoptosis, but this result suggested that Bax might also play a role in necrosis. To determine if this was the case, we engineered Bax mutants that lack its different functional domains. We studied the ability of each mutant protein to regulate apoptosis and necrosis in the zebrafish and found the BH3 domain, which is required for Bax oligomerization during apoptosis, not to be required for necrosis under excess TNF conditions. Rather, an C-terminal transmembrane helix was found to be required for necrosis. These findings demonstrate that the pro-apoptotic factor Bax also regulates necrosis but through a distinct functional domain. Since excess TNF-mediated necrosis has been implicated in human TB pathogenesis, this understanding of how Bax regulates necrosis may guide host-derived therapies.

Tuberculosis (TB) is a bacterial infection that leads to 3 million deaths per year worldwide. Proper control of TB requires the ability of patrolling macrophages to detect, engulf and destroy invading bacteria and it is currently understood that macrophages play a key role in responding to early Mycobacterial infection. A forward genetic screen using the zebrafish larval model for host determinants of immunity to the bacterium Mycobacterium marinum found a hypersusceptible mutant that contains a nonsense mutation in the mTor (mechanistic target of rapamycin) gene. mTor has been shown in other systems to be crucial to protein synthesis, cell growth, and survival. Currently, our work has shown that mTor mutant fish have fewer macrophages and neutrophils than their wild type siblings. We have also shown that mTor mutants display hypersusceptibility to M. marinum infection, with bacteria displaying exuberant extracellular growth in the fish.. Treatment of fish with Rapamycin, an mTor inhibitor, phenocopies the macrophage deficiency and hypersusceptibility observed in the mutant. We hypothesized that hypersusceptibility to M. marinum in mTor mutant zebrafish is primarily due to the deficiency of macrophage immune cells. To test this hypothesis, we depleted macrophages in wild-type and mutant fish using lipoclodronate. Preliminary data has shown that without macrophages, mTor mutants still exhibit increased infection burden compared to wild type siblings also treated with lipoclodronate. This data provides us with exciting new avenues to continue searching for the cause of mTor’s hypersusceptibility. We are pursuing mTor’s role in neutrophil and cytokine induction as additional explanations for the mTor mutant hypersusceptibility phenotype. This research will help define the innate immune response to Mycobacterial infection and clarify the respective functions of mTor in host immunity.