Mechanisms of Type III Secretion System ATPase Activation and Regulation

III型分泌系统ATP酶激活及调节机制

• 批准号: 9231935
• 负责人: Nicholas E Dickenson
• 金额: $ 41.14万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-23 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231935
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Affect; Anti-Infective Agents; Architecture; Bacillary Dysentery; Bacteria; Binding; Biological Assay; Biological Models; Cell physiology; Cells; Cessation of life; Child; Comparative Study; Complex; Cytoplasm; Data; Development; Energy-Generating Resources; Engineering; Enzyme Activation; Enzymes; Fluorescence; Fluorescence Anisotropy; Fluorescent Probes; Homo; Human; Immune response; Individual; Infection; Injectable; Knowledge; Lead; Link; Measurement; Methods; N-terminal; Needles; Nucleotides; Pathogenesis; Phenylalanine; Play; Positioning Attribute; Prevention; Proteins; Recombinant Proteins; Recombinants; Regulation; Resolution; Role; Salmonella; Sequence Homology; Shigella; Shigella Infections; Shigella flexneri; Site; Structure; System; Techniques; Testing; Thermodynamics; Type III Secretion System Pathway; Virulence; Virulence Factors; Work; base; biophysical properties; enzyme activity; experimental study; genetic analysis; genetic regulatory protein; human disease; inhibitor/antagonist; insight; interdisciplinary approach; monomer; mutant; novel; pathogen; pathogenic bacteria; public health relevance; tool; trait; unnatural amino acids

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.

项目总结/摘要 福氏志贺菌是细菌性痢疾的病原体，它利用其III型分泌系统（T3 SS）作为管道 通过该通道效应蛋白从细菌穿梭到宿主细胞质。一旦被注射，它们就会颠覆 正常的宿主功能和促进感染和防御宿主免疫反应。虽然很多关于 T3 SS功能已制定，如何控制系统组装和激活的详细信息尚未确定 很好理解。志贺氏菌蛋白Spa 47和已知ATP酶之间的序列同源性表明， 可以通过ATP水解机制提供必要的能量。我们假设志贺氏菌利用这个 ATP酶激活和控制T3 SS并最终感染人类宿主细胞的能力。这项建议 描述了最近的发展，提供了第一个成功的表达和纯化的ATP酶活性 spa 47和纯化酶的生物物理特性。这些初步发现提供了一个强有力的平台， 有针对性的研究表明，Spa 47的激活是通过形成前所未有的T3 SS来控制的。 ATP酶同源三聚体，可能是由于缺乏N-末端序列（沙门氏菌共有）所致 ATP酶InvC），但在T3 SS ATP酶中是保守的。此外，在此基础上进行的实验 知识鉴定志贺氏菌蛋白MxiN作为Spa 47活性的有效抑制剂，暗示它是关键的 志贺氏菌T3 SS的调节组分。为了更好地了解Spa 47在志贺菌T3 SS中的调节作用， 相关的感染，并确定这种潜在的新型T3 SS ATP酶亚类的特征，特异性 本研究的目的是：1）使用福氏志贺菌作为模型系统，以阐明 Spa 47寡聚化、ATP水解活性和T3 SS相关毒力。（2）表征 Spa 47和新鉴定的志贺氏菌T3 SS调节蛋白MxiN之间的调节相互作用。 拟议的研究将采用跨学科的方法，直接观察Spa 47在 志贺氏菌的背景以及确定寡聚化和MxiN相互作用对活化的影响 以及调节Spa 47的ATP酶活性。这些发现将弥合人们在理解 包括志贺氏菌和沙门氏菌在内的一类重要的人类病原体是如何通过 T3 SS激活的适当时机。此外，这些研究将揭示Spa 47的机制细节。 该活性可导致开发用于更有效地治疗感染的化合物和方法， 表达T3 SS的病原体。