Understanding the mechanisms of ESX secretion systems in mycobacteria

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PROJECT SUMMARY Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that takes the lives of millions of people annually. With the rise of multidrug resistant mycobacteria there is a dire need for development of new antibiotics and therapies. The ESX-3 secretion system is an attractive target, as it is required for the selection and release of various M. tuberculosis substrates and is required for iron acquisition and homeostasis. This latter role is essential in M. tuberculosis, such that if ESX-3 is knocked out the bacteria will not survive in vitro without excess iron supplementation. Development of a new therapeutic, such as a small molecule that prevents the secretion mechanism underpinning the function of the complex, requires an understanding of the ESX-3 structure. The Rosenberg lab has determined the structure of the model organism M. smegmatis ESX-3 secretion system using cryo-electron microscopy. M. smegmatis is a nonpathogenic, fast growing mycobacterium that expresses ESX proteins homologous to M. mycobacterium. The cryo-EM structure has provided novel insight into how ESX-3 complexes oligomerize, as well as how the individual components that comprise the ESX-3 complex interact with one another. Based on our structure, which we believe is the complex trapped in the "off" state, I hypothesize that substrate secretion through ESX-3 is modulated by changes in the molecular configuration of the machine. In Aim 1, I will use a combination of mycobacterial genetics, structural biology, and cryo-EM to trap the ESX-3 complex in an alternative, active state that will inform how the complex rearranges during secretion. In Aim 2, I will use deep mutational scanning and a survival assay I developed to further investigate the importance and function of the core ESX-3 proteins using the previously mentioned low- iron growth phenotype, where if an essential protein, protein domain, or protein residue is mutated, the bacteria will not survive in chelated iron media. Taken together, these aims will allow me to study the ESX-3 secretion mechanism and determine which intra-complex interactions are required to facilitate this mechanism. These studies may identify new targets to develop novel antibiotics and might also provide a better understanding of the cell biology of mycobacteria.