Understanding the mechanisms of ESX secretion systems in mycobacteria

项目摘要 结核分枝杆菌是结核病的病原体，结核病是一种夺去数百万人生命的疾病。 人每年。随着多药耐药分枝杆菌的增加，迫切需要开发新的抗结核药物。 抗生素和治疗。ESX-3分泌系统是一个有吸引力的目标，因为它是选择所需的。 和释放各种M.结核病的底物和所需的铁的收购和稳态。后一 在M.结核病，使得如果ESX-3被敲除，细菌将不能在体外存活， 过量补铁开发一种新的治疗方法，如一种小分子， 支持复合物功能的分泌机制，需要了解ESX-3 结构罗森伯格实验室已经确定了模式生物M的结构。斯美拉ESX-3 使用冷冻电子显微镜观察分泌系统。M.包皮垢病是一种非致病性的，快速生长的 表达与分枝杆菌同源的ESX蛋白的分枝杆菌。分枝杆菌该冷冻EM结构具有 提供了新的见解ESX-3复合物如何寡聚化，以及如何单独的组件， 包括ESX-3复合体相互作用。根据我们的结构，我们认为这是复杂的 在“关闭”状态下，我假设通过ESX-3的底物分泌是由 机器的分子结构在目标1中，我将使用分枝杆菌遗传学、结构 生物学和cryo-EM将ESX-3复合体捕获在另一种活跃的状态下， 在分泌过程中重新排列。在目标2中，我将使用深度突变扫描和我开发的存活测定， 进一步研究核心ESX-3蛋白的重要性和功能，使用先前提到的低- 铁生长表型，其中如果必需蛋白质、蛋白质结构域或蛋白质残基突变，则细菌 不能在螯合铁介质中存活综上所述，这些目标将使我能够研究ESX-3分泌 机制，并确定需要哪些复合体内的相互作用来促进这种机制。这些 研究可能会发现新的目标，以开发新的抗生素，也可能提供更好的理解， 分枝杆菌的细胞生物学

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.