Project Summary / Abstract: Due to the rise of antibiotic resistant tuberculosis and the long treatment time of current anti-tuberculosis therapies, there is an immediate need for new drug targets against tuberculosis. My lab has identified the mycobacterial BrkB ortholog as a likely drug target. We have already observed that mutation of mycobacterial BrkB greatly attenuates growth of our model organism Mycobacterium marinum in its natural host, the zebrafish. Furthermore, we have observed that this organism is attenuated for growth in both intracellular and extracellular spaces, making it particularly attractive because extracellular mycobacteria are especially present in late-stage TB and contribute to TB transmission, and because there is a specific need to target mycobacteria in the extracellular niche. While BrkB is found throughout the bacterial kingdom, the function of BrkB remains elusive. I propose to characterize the biochemical function of mycobacterial BrkB and resolve its role in virulence by completing the following three specific aims: (1) Identify mycobacterial BrkB channel substrate(s) and test for protein interactions, (2) identify mechanism of BrkB extracellular growth requirement, and (3) Measure mycobacterial BrkB growth under late-stage infection conditions. Towards this end, I have purified recombinant tuberculosis BrkB from E. coli. In collaboration with Dr. Paul DeCaen (Northwestern Univ.), my students and I will test BrkB for channel function. My lab has extensive experience with the biochemistry, microbiology, and organismal biology required to complete aims 1,2, and 3 respectively. With the biophysical expertise of the DeCaen lab, we collectively share the expertise to solve the role of BrkB in the mycobacterial cell and in the context of infection. This project will serve as the foundation of a sustainable research platform that will empower undergraduates to test independent hypotheses about mycobacterial virulence factors and host susceptibility factors. Students will have the tools necessary to investigate the biochemical functions of these functions, to work at biosafety level 2 to investigate mycobacterial cellular biology, and to test their molecular hypotheses at the organismal level using the zebrafish Mycobacterium marinum model of infection.