Functional characterization of HtrA, an essential mycobacterial protease

项目概要/摘要 结核病是全球死亡的主要原因，感染了全球1/3的人口。但 分枝杆菌细胞壁的复杂化学组成，结核分枝杆菌的强大弹性 (Mtb)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的 治疗这种疾病。分枝杆菌蛋白酶，虽然研究不足，代表了一类潜在的新的 药物靶点，因为它们的保守性和对细胞生理学的重要性。虽然高度 在大多数其他细菌中，保守的丝氨酸蛋白酶HtrA是生长必需的，Mtb似乎需要HtrA 在多种背景下的活动。总的来说，这项建议旨在确定HtrA的各种职能如何发挥作用， 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。要求1 询问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域的功能。在这一目标中，我将评估HtrA独特的细胞质中的贡献， 结构域及其基于丝氨酸的蛋白水解活性，使用遗传学和生物化学的组合来鉴定 HtrA的结合伴侣和底物。目的2研究HtrA在细胞周期调控中的非蛋白水解作用， 使用多种互补策略来鉴定与HtrA相互作用的细胞壁酶， HtrA发挥作用的细胞周期途径。为了实现这一点，我将验证假定的HtrA结合伙伴， 细胞壁界面，用延时显微镜跟踪HtrA动力学，并揭示HtrA的遗传相互作用 特定途径的药物。总之，这些目标在两个不同的背景下定义了HtrA-作为一种蛋白酶至关重要 用于对抗环境压力和作为新的细胞周期调节剂。这项工作将提供深入了解 分枝杆菌HtrA具有前所未有的重要性和多样的功能， 控制结核菌的生长和生存最终，这项工作有可能填补知识的巨大空白 Mtb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.