Manipulation of the actin cytoskeleton by spotted fever group Rickettsia

斑疹热立克次体对肌动蛋白细胞骨架的操纵

• 批准号: 7297373
• 负责人: Matthew D Welch
• 金额: $ 37.75万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7297373
• 关键词: Actins; Animal Model; Architecture; Bacteria; Biochemical; Biological; Cell physiology; Cells; Complement; Complex; Cytoskeletal Proteins; Cytoskeleton; Diagnosis; Drosophila melanogaster; Electron Microscopy; Fever; Filament; Fluorescence Microscopy; Fractionation; Human; Infection; Legionella pneumophila; Light; Listeria monocytogenes; Longitudinal Studies; Microfilaments; Modeling; Molecular; Movement; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Play; Process; Proteins; RNA Interference; Research Personnel; Rickettsia; Rocky Mountain Spotted Fever; Role; Spottings; Staging; Surface; System; Tail; Testing; Time; Transmembrane Domain; Type IV Secretion System Pathway; base; cell motility; insight; interest; member; novel strategies; pathogen; polymerization; programs

DESCRIPTION (provided by applicant): Members of the spotted fever group (SFG) of the genus Rickettsia are obligate intracellular bacteria that cause serious human illnesses such as Rocky Mountain Spotted Fever. They require the host actin cytoskeleton to facilitate internalization into host cells, and to power intracytoplasmic movement that enables spread between cells during infection. Therefore, elucidating how SFG Rickettsia manipulate the actin cytoskeleton is critical for understanding the process of pathogenesis. In addition, determining the mechanism of actin-based motility is of great interest from a cell biological perspective because actin filaments in Rickettsia comet tails differ considerably in their organization compared to filaments assembled by well-studied pathogens such as Listeria monocytogenes, suggesting that Rickettsia use different molecules and mechanisms to promote motility. Despite the importance of actin in entry and motility, very little is known about the molecular mechanisms used by the bacteria to manipulate the cytoskeleton. We recently identified a Rickettsia protein called RickA that is conserved among SFG species and stimulates actin polymerization by activating the host Arp2/3 complex, providing the first molecular insight into how Rickettsia harness actin. We now need to answer important questions about the role of RickA and Arp2/3 in infection. For example, do RickA and Arp2/3 complex play a functionally important role, and how to they act? When and how is RickA introduced into host cells to stimulate actin polymerization? What other cytoskeletal proteins are required to polymerize and organize actin filaments? Based our preliminary results, we propose a unifying hypothesis that RickA is translocated by bacteria into host cells using a type IV secretion system, where it activates Arp2/3 complex to initiate actin assembly during entry and/or motility. We further hypothesize that, during actin-based motility, RickA and Arp2/3 act transiently, and other cyoskeletal proteins are subsequently needed to generate the unique organization of actin filaments in comet tails. To test this hypothesis, we propose the following aims: (1) Determine the timing and localization of the secreted RickA protein and the mechanism of secretion, (2) Examine the function of Arp2/3 complex during entry and actin- based motility, and (3) test the role of the full spectrum of actin cytoskeletal proteins in entry and motility. Determining the mechanisms used by SFG Rickettsiae to manipulate the host actin cytoskeleton will shed light on an essential and poorly understood aspect of Rickettsia pathogenesis, and will also illuminate the mechanisms used by host cells to regulate the functions of the cytoskeleton. In the long-term these studies may result in the discovery of new paradigms for understanding host-pathogen interactions, and new approaches to diagnose and treat infections.

描述（由申请人提供）：立克次体属斑点热组（SFG）的成员是专性细胞内细菌，可引起严重的人类疾病，如落基山斑点热。它们需要宿主肌动蛋白细胞骨架来促进内化到宿主细胞中，并为胞质内运动提供动力，从而在感染期间在细胞之间传播。因此，阐明SFG立克次体如何操纵肌动蛋白细胞骨架对于理解致病过程至关重要。此外，从细胞生物学的角度来看，确定肌动蛋白为基础的运动机制是非常感兴趣的，因为在立克次体彗星尾巴肌动蛋白丝在其组织中有很大的不同相比，由充分研究的病原体，如单核细胞增生李斯特菌组装的细丝，这表明立克次体使用不同的分子和机制，以促进运动。尽管肌动蛋白在进入和运动中的重要性，但对细菌操纵细胞骨架的分子机制知之甚少。我们最近发现了一种称为RickA的立克次体蛋白，该蛋白在SFG物种中是保守的，并通过激活宿主Arp 2/3复合物来刺激肌动蛋白聚合，这为立克次体如何利用肌动蛋白提供了第一个分子见解。我们现在需要回答关于RickA和Arp 2/3在感染中的作用的重要问题。例如，RickA和Arp 2/3复合体是否在功能上起重要作用，以及它们是如何起作用的？RickA何时以及如何被引入宿主细胞以刺激肌动蛋白聚合？还有哪些其他的细胞骨架蛋白质是固定和组织肌动蛋白丝所必需的？基于我们的初步结果，我们提出了一个统一的假设，RickA是易位的细菌进入宿主细胞使用IV型分泌系统，在那里它激活Arp 2/3复合物启动肌动蛋白组装在进入和/或运动。我们进一步假设，在肌动蛋白为基础的运动，RickA和Arp 2/3的行为短暂，和其他cyoskeleton蛋白随后需要产生独特的组织肌动蛋白丝的彗星尾巴。为了验证这一假设，我们提出了以下目标：（1）确定分泌的RickA蛋白的时间和定位以及分泌机制，（2）检查Arp 2/3复合物在进入和基于肌动蛋白的运动过程中的功能，以及（3）测试全谱肌动蛋白细胞骨架蛋白在进入和运动中的作用。确定SFG立克次体操纵宿主肌动蛋白细胞骨架的机制，将揭示立克次体发病机制的一个重要而又知之甚少的方面，也将阐明宿主细胞调节细胞骨架功能的机制。从长远来看，这些研究可能会发现理解宿主-病原体相互作用的新范例，以及诊断和治疗感染的新方法。