Mechanisms of Type III Secretion System ATPase Activation and Regulation

项目摘要/摘要 细菌性痢疾的病原体福氏志贺氏菌利用其III型分泌系统(T3SS)作为管道 通过它，效应蛋白从细菌穿梭到宿主细胞细胞质。一旦注射，它们就会颠覆 正常的宿主功能，促进感染和防御宿主的免疫反应。虽然有很多关于 已经制定了T3SS功能，但没有详细说明如何控制系统组装和激活 很好理解。志贺氏菌蛋白Spa47与已知ATPase的序列同源性表明，它 可能通过三磷酸腺苷的水解机制提供必要的能量。我们假设志贺氏菌使用这种 ATPase激活和控制T3SS，最终感染人类宿主细胞的能力。这项建议 描述了首次成功表达和纯化ATPase活性的最新进展 Spa47和纯化的酶的生物物理性质。这些初步发现提供了一个强大的平台 定向研究表明，Spa47的激活是通过形成前所未有的T3SS来控制的 可能由缺乏N-末端序列(沙门氏菌共有的)驱动的ATPase同源三聚体 ATPase InvC)，但在其他方面在T3SS ATPase中保守。此外，在此基础上进行的实验 知识发现志贺氏菌蛋白MxiN是Spa47活性的有效抑制物，这意味着它是一个关键 志贺氏菌T3SS的调节成分。为了更好地了解Spa47在志贺氏菌T3SS中的调节作用- 相关感染，并定义这一潜在的新的T3SS ATPase亚类的特征，特异性 本研究的目的是：1)以福氏志贺氏菌为模型系统，研究福氏志贺氏菌与福氏志贺氏菌之间的关系 Spa47寡聚、ATP水解酶活性和T3SS相关毒力。和2)描述 Spa47与新发现的志贺氏菌T3SS调控蛋白MxiN之间的调控相互作用。 拟议的研究将采用跨学科的方法来直接观察SPA47在 志贺氏菌的背景以及寡聚和MxiN相互作用对激活的影响 以及对Spa47的ATPase活性的调节。这些发现将弥合人们在理解上的一个重大差距 包括志贺氏菌和沙门氏菌在内的一类重要的人类病原体如何通过 T3SS激活的适当时机。此外，这些研究还将揭示Spa47的机制细节 可导致开发化合物和更有效地通过以下方式治疗感染的方法的活动 表达T3SS的病原菌。

Project Summary/Abstract Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (T3SS) as a conduit through which effector proteins are shuttled from bacterial to host cell cytoplasm. Once injected, they subvert normal host functions and promote infection and defend against host immune responses. While much about T3SS function has been worked out, details of how the system assembly and activation are controlled are not well understood. Sequence homology between the Shigella protein Spa47 and known ATPases suggests that it may provide the necessary energy through ATP hydrolysis mechanisms. We hypothesized that Shigella use this ATPase to activate and control the T3SS and ultimately the ability to infect human host cells. This proposal describes recent developments that provided the first successful expression and purification of ATPase active Spa47 and biophysical characterization of the purified enzyme. These initial findings provided a strong platform for directed studies showing that Spa47 activation is controlled through the formation of an unprecedented T3SS ATPase homo-trimer that may be driven by the lack of an N-terminal sequence (shared by the Salmonella ATPase InvC) but otherwise conserved amongst T3SS ATPases. Additionally, experiments building on this knowledge identified the Shigella protein MxiN as a potent inhibitor of Spa47 activity, implicating it as a key regulatory component of the Shigella T3SS. To better understand the regulatory role of Spa47 in Shigella T3SS- associated infection and to define the traits of this potentially novel sub-class of T3SS ATPases, the specific aims of this study are to: 1) Use Shigella flexneri as a model system to unravel the relationship between Spa47 oligomerization, ATP hydrolysis activity, and T3SS-associated virulence. and 2) Characterize regulatory interactions between Spa47 and the newly identified Shigella T3SS regulatory protein MxiN. The proposed studies will employ an interdisciplinary approach to directly observe the effects of Spa47 within the context of Shigella as well as determine the influence of oligomerization and MxiN interaction on activation and regulation of the ATPase activity of Spa47. These findings will close a significant gap in the understanding of how an important class of human pathogens including Shigella and Salmonella control their virulence through appropriate timing of T3SS activation. Additionally, these studies will uncover mechanistic details of Spa47 activity that could lead to the development of compounds and methods for more efficiently treating infections by T3SS-expressing pathogens.