Functional characterization of HtrA, an essential mycobacterial protease

项目概要/摘要 结核病是全球主要死亡原因，感染了全球 1/3 的人口。然而， 分枝杆菌细胞壁化学成分复杂，结核分枝杆菌抵抗力强 （结核分枝杆菌）在恶劣的宿主环境中，不断增加的抗生素耐药性阻碍了有效 治疗这种疾病。分枝杆菌蛋白酶虽然尚未得到充分研究，但却代表了一类潜在的新产品 药物靶点因其高度保守的性质和对细胞生理学的重要性。虽然高度 保守的丝氨酸蛋白酶 HtrA 对于大多数其他细菌的生长是可有可无的，结核分枝杆菌似乎需要 HtrA 多个上下文中的活动。从广义上讲，该提案旨在定义 HtrA 的各种功能如何做出贡献 1) 结核分枝杆菌对环境压力的反应和 2) 分枝杆菌细胞周期的调节。目标1 探讨 HtrA 在分枝杆菌应激反应中的作用，采用遗传学方法来描述 每个 HtrA 结构域的功能。为此，我将评估 HtrA 独特的细胞质的贡献 结构域及其基于丝氨酸的蛋白水解活性，利用遗传学和生物化学的结合来识别 HtrA 的结合伴侣和底物。目标 2 研究 HtrA 在细胞周期调节中的非蛋白水解作用， 使用多种互补策略来识别与 HtrA 相互作用的细胞壁酶并定义 HtrA 发挥作用的细胞周期途径。为了实现这一目标，我将验证假定的 HtrA 结合伙伴： 细胞壁界面，通过延时显微镜追踪 HtrA 动力学，并揭示 HtrA 的遗传相互作用 与途径特异性药物。这些目标共同在两种不同的背景下定义了 HtrA——作为一种关键的蛋白酶 用于对抗环境压力并作为新型细胞周期调节剂。这项工作将提供对 分枝杆菌HtrA前所未有的重要性和多样化功能，以及独特的机制 控制 Mtb 的生长和生存。最终，这项工作有可能填补巨大的知识空白 Mtb 在人类感染中的持续存在，并为抗分枝杆菌药物提供了新的靶点。

Project Summary/Abstract Tuberculosis is a leading cause of death worldwide, infecting 1/3 of the global population. However, the complex chemical composition of the mycobacterial cell wall, robust resilience of Mycobacterium tuberculosis (Mtb) in harsh host environments, and ever-increasing rates of antibiotic resistance have hindered effective treatment of this disease. Mycobacterial proteases, while understudied, represent a class of potential novel drug targets due to their well-conserved nature and importance to cellular physiology. Although the highly conserved serine protease HtrA is dispensable for growth in most other bacteria, Mtb appears to require HtrA activity in multiple contexts. Broadly, this proposal seeks to define how the various functions of HtrA contribute to 1) Mtb's response to environmental stress and 2) the regulation of the mycobacterial cell cycle. Aim 1 interrogates HtrA's role in the mycobacterial stress response, taking a genetic approach to delineate the functions of each of HtrA's domains. In this aim, I will assess the contributions of HtrA's unique cytoplasmic domain and its serine-based proteolytic activity, using a combination of genetics and biochemistry to identify HtrA's binding partners and substrates. Aim 2 investigates HtrA's non-proteolytic role in cell cycle regulation, using multiple complementary strategies to identify cell wall enzymes with which HtrA interacts and define the cell cycle pathways in which HtrA operates. To accomplish this, I will validate putative HtrA binding partners at the cell wall interface, track HtrA kinetics with time-lapse microscopy, and uncover HtrA's genetic interactions with pathway-specific drugs. Together, these aims define HtrA in two different contexts – as a protease crucial for combating environmental stress and as a novel cell cycle regulator. This work will provide insight into the unprecedented essentiality and diverse functions of mycobacterial HtrA, as well as the unique mechanisms that govern the growth and survival of Mtb. Ultimately, this work has the potential to fill large gaps in knowledge of Mtb's persistence in the context of human infection and provide novel targets for antimycobacterial drugs.