Functional characterization of HtrA, an essential mycobacterial protease

项目摘要/摘要 结核病是全球主要的死亡原因，感染了全球三分之一的人口。然而， 结核分枝杆菌细胞壁化学成分复杂，抗结核能力强 (MTB)在恶劣的宿主环境中，不断增加的抗生素耐药率阻碍了有效的 治疗这种疾病的方法。分枝杆菌蛋白水解酶虽然没有得到充分的研究，但却代表了一类潜在的新的 药物靶标，因为它们的保守性很好，对细胞生理学很重要。虽然高度的 保守的丝氨酸蛋白酶HtrA对于大多数其他细菌的生长是必不可少的，Mtb似乎需要HtrA 在多个上下文中的活动。概括地说，这项建议旨在定义HtrA的各种职能如何贡献 对1)结核分枝杆菌对环境胁迫的反应；2)对分枝杆菌细胞周期的调节。目标1 询问htrA在分枝杆菌应激反应中的作用，采用遗传方法描述 HtrA的每个域的功能。在这个目标中，我将评估htrA独特的细胞质的贡献 结构域及其丝氨酸蛋白水解性，使用遗传学和生物化学相结合的方法来鉴定 HtrA的结合伙伴和底物。目的2研究htrA在细胞周期调控中的非蛋白降解作用。 使用多种互补策略来确定HtrA与之相互作用的细胞壁酶并定义 HtrA作用的细胞周期途径。为此，我将在以下位置验证假定的HtrA结合伙伴 细胞壁界面，用延时显微镜跟踪HtrA的动力学，并揭示HtrA的遗传相互作用 使用特定途径的药物。总而言之，这些目标定义了HtrA在两种不同的背景下-作为一种至关重要的蛋白酶 用于对抗环境压力和作为一种新型的细胞周期调节因子。这项工作将提供对 分枝杆菌HtrA前所未有的重要性和多样的功能以及独特的机制 管理着结核分枝杆菌的成长和生存。归根结底，这项工作有可能填补知识的巨大空白 研究结核分枝杆菌在人类感染背景下的持久性，并为抗分枝杆菌药物提供新的靶点。

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.