Understanding the mechanisms of ESX secretion systems in mycobacteria

项目总结 结核分枝杆菌是肺结核的病原体，这种疾病夺走了数百万人的生命 每年都有人。随着耐多药分支杆菌的兴起，迫切需要开发新的 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 和释放各种结核分枝杆菌底物，是铁的获取和动态平衡所必需的。后者 在结核分枝杆菌中起着至关重要的作用，如果ESX-3基因被敲除，细菌将无法在体外存活，除非 补充过多的铁。开发一种新的治疗方法，例如一种小分子，可以防止 支持该复合体功能的分泌机制，需要了解ESX-3 结构。罗森博格实验室已经确定了模式生物污垢分枝杆菌ESX-3的结构 用冷冻电子显微镜观察分泌系统。污垢分枝杆菌是一种非致病性的、生长迅速的 表达与分枝杆菌同源的ESX蛋白的分枝杆菌。低温电磁结构具有 提供了对ESX-3复合体如何齐聚以及单个组件如何进行齐聚的新见解 组成ESX-3复合体相互作用。基于我们的结构，我们认为这是复杂的 处于“关闭”状态时，我推测通过ESX-3分泌的底物受 机器的分子配置。在目标1中，我将使用分枝杆菌遗传学、结构性 生物学和冷冻-EM将ESX-3复合体捕获在另一种活跃状态，这将告知复合体如何 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生存测试 进一步研究核心ESX-3蛋白的重要性和功能，使用前面提到的低- 铁生长表型，如果必需的蛋白质、蛋白质结构域或蛋白质残基发生突变，细菌 将不会在螯合铁介质中存活。总而言之，这些目标将使我能够研究ESX-3的分泌 机制，并确定需要哪些复杂的内部交互来促进这一机制。这些 研究可能确定开发新抗生素的新靶点，也可能提供更好的了解 分支杆菌的细胞生物学。

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.