Manipulation of the actin cytoskeleton by spotted fever group Rickettsia

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宿主细胞内化，并为感染期间细胞间传播提供动力。因此，阐明SFG立克次体如何操纵肌动蛋白细胞骨架对于了解其发病过程至关重要。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有趣的，因为立克次体彗星尾部的肌动蛋白丝与单核增生李斯特菌等病原体聚集的丝在组织上有很大的不同，这表明立克次体使用不同的分子和机制来促进运动。尽管肌动蛋白在进入和运动中很重要，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名为RickA的立克次体蛋白，该蛋白在SFG物种中保守，并通过激活宿主Arp2/3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Arp2/3在感染中的作用的重要问题。例如，RickA和Arp2/3复合体是否在功能上起重要作用，它们是如何起作用的？何时以及如何将RickA引入宿主细胞以刺激肌动蛋白聚合？聚合和组织肌动蛋白丝需要哪些其他细胞骨架蛋白？基于我们的初步结果，我们提出了一个统一的假设，即细菌通过IV型分泌系统将RickA转运到宿主细胞中，在进入和/或运动期间激活Arp2/3复合体启动肌动蛋白组装。我们进一步假设，在基于肌动蛋白的运动过程中，RickA和Arp2/3是短暂的，随后需要其他细胞骨骼蛋白来产生彗星尾巴中独特的肌动蛋白细丝组织。为了验证这一假设，我们提出了以下目标：(1)确定RickA蛋白分泌的时间和位置以及分泌机制；(2)检查Arp2/3复合物在进入和基于肌动蛋白的运动中的功能；(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基本和鲜为人知的方面，也将阐明宿主细胞调节细胞骨架功能的机制。从长远来看，这些研究可能会导致发现理解宿主-病原体相互作用的新范式，以及诊断和治疗感染的新方法。

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.