Rickettsia mobilization of the cytoskeleton during invasion, motility, and spread

描述（由申请人提供）：立克次体是专性细胞内细菌病原体，可引起斑疹热和斑疹伤寒等疾病。我们研究了引起人类立克次体病的斑点热组（SFG）模型物种立克次体（Rickettsia parkeri），该物种实验易感，并且具有小鼠发病模型，为揭示SFG立克次体感染的分子机制和毒力提供了理想的方法。与单核增生李斯特菌等病原体类似，SFG立克次体需要宿主肌动蛋白细胞骨架侵入，在宿主细胞内移动并在宿主细胞之间传播。然而，立克次体在操纵肌动蛋白的机制上不同于其他病原体。因此，研究立克次体将促进我们对病原体如何以及为什么利用不同的肌动蛋白聚合机制的理解，并将揭示未感染细胞中肌动蛋白功能和调控的新特征。我们过去的工作对理解立克次体如何动员肌动蛋白做出了重要贡献。我们发现了介导入侵的宿主细胞骨架蛋白的核心集合，并单独揭示了对立克次体细胞内运动独特重要的宿主蛋白。此外，我们发现立克次体在使用两个连续的运动阶段上与其他病原体不同——依赖于细菌RickA蛋白的早期阶段和依赖于细菌Sca2蛋白的晚期阶段。尽管取得了这些进展，但人们对立克次体在感染期间如何动员细胞骨架知之甚少。利用最近分离的一组parkeri转座子突变体，我们将测试立克次体部署包括Sca2在内的蛋白质，它们的IV型分泌系统（T4SS），假定的T4SS效应物和肌动蛋白相关蛋白RickA和Sca4，以及锚蛋白重复蛋白Ank1和Ank2，在宿主细胞入侵，细胞内运动和细胞-细胞传播过程中动员肌动蛋白的整体假设。我们将回答三个问题。首先，细菌蛋白在入侵过程中是如何促进肌动蛋白组装的？其次，立克次体如何以及为什么使用两种肌动蛋白组装因子来实现细胞内运动的不同阶段？第三，细菌蛋白如何促进细胞-细胞扩散？更具体地说，在Aim 1中，我们将测试立克次体是否以及如何在入侵和毒力中使用Sca2、T4SS和假定的T4SS效应物Sca4、RickA和Ank2，并确定这些因素是否为T4SS效应物。在Aim 2中，我们将测试早期和晚期运动是否促进了自噬对细菌杀伤的避免，确定早期和晚期运动发生的时间和方式，并检查RickA和Sca2介导运动和毒力的分子机制。在Aim 3中，我们将确定早期和晚期运动在细胞-细胞扩散中的作用，并研究T4SS、RickA、Sca4、Ank1和Ank2在不同扩散阶段调节肌动蛋白和膜动力学的重要性。这些研究将揭示宿主-病原体相互作用的新机制，并可能产生诊断和治疗感染的新方法。

DESCRIPTION (provided by applicant): Rickettsiae are obligate intracellular bacterial pathogens that cause diseases such as spotted fever and typhus. We study the model spotted fever group (SFG) species Rickettsia parkeri, which causes human rickettsiosis, is experimentally tractable, and has a mouse model of pathogenesis, making it ideal for revealing molecular mechanisms of SFG Rickettsia infection and virulence. Similar to pathogens such as Listeria monocytogenes, SFG Rickettsia require the host actin cytoskeleton to invade, move within, and spread between host cells. However, Rickettsia are distinct from other pathogens in their mechanisms of manipulating actin. Therefore, studying Rickettsia will advance our understanding of how and why pathogens exploit diverse actin-polymerizing mechanisms, and will reveal new features of actin function and regulation in uninfected cells. Our past work has made important contributions to understanding how Rickettsia mobilize actin. We uncovered a core set of host cytoskeletal proteins that mediate invasion, and separately revealed host proteins uniquely important for Rickettsia intracellular motility. Moreover, we discovered that Rickettsia are distinct from other pathogens in using two sequential phases of motility - an early phase that depends on the bacterial RickA protein, and a late phase that depends on the bacterial Sca2 protein. Despite these advances, how Rickettsia mobilize the cytoskeleton during infection is poorly understood. Using a panel of recently isolated transposon mutants of R. parkeri, we will test the overall hypothesis that Rickettsia deploy proteins including Sca2, their type IV secretion system (T4SS), the putative T4SS effectors and actin-associated proteins RickA and Sca4, as well as ankyrin-repeat proteins Ank1 and Ank2, to mobilize actin during host cell invasion, intracellular motility and cell-cell spread. We will answer three questions. First, how do bacterial proteins promote actin assembly during invasion? Second, how and why do Rickettsia use two actin assembly factors to enable distinct phases of intracellular motility? Third, how do bacterial proteins contribute to cell-cell spread? More specifically, in Aim 1, we will test whether and how Rickettsia use Sca2, the T4SS, and the putative T4SS effectors Sca4, RickA and Ank2 in invasion and virulence, and determine if these factors are T4SS effectors. In Aim 2, we will test whether early and late motility promote avoidance of bacterial killing by autophagy, determine when and how a transition between early and late motility occurs, and examine the molecular mechanisms by which RickA and Sca2 mediate motility and virulence. In Aim 3, we will determine the role of early and late motility in cell-cell spread, and examine the importance of the T4SS, RickA, Sca4, Ank1 and Ank2 in modulating actin and membrane dynamics at distinct stages of spread. These studies will reveal new mechanisms of host-pathogen interactions, and may result in new ways of diagnosing and treating infections.