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

描述(申请人提供)：立克次体是引起斑点热和斑疹伤寒等疾病的主要细胞内细菌病原体。我们研究了引起人类立克次体病的模式斑点热群(SFG)种帕氏立克次体(Rickettsia Parkeri)，它在实验上容易处理，并具有小鼠致病模型，使其成为揭示SFG立克次体感染和毒力的分子机制的理想工具。与单核细胞增多性李斯特氏菌等病原体类似，SFG立克次体需要宿主肌动蛋白细胞骨架入侵、在宿主细胞内移动和在宿主细胞之间传播。然而，立克次体在其操纵肌动蛋白的机制上与其他病原体不同。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。我们发现了一组介导入侵的核心宿主细胞骨架蛋白，并分别揭示了宿主蛋白对立克次体细胞内运动的独特重要性。此外，我们发现立克次体不同于其他病原体，它使用两个连续的运动阶段-早期依赖于细菌RickA蛋白，晚期依赖于细菌sca2蛋白。尽管取得了这些进展，但立克次体如何在感染过程中动员细胞骨架仍鲜为人知。利用最近分离的帕氏杆菌转座子突变体，我们将检验这一总体假设，即立克次体在宿主细胞入侵、细胞内运动和细胞-细胞扩散过程中部署包括ScA2及其IV型分泌系统(T4SS)、假定的T4SS效应蛋白和肌动蛋白相关蛋白RickA和Sca4以及Ank1和Ank2在内的蛋白来动员肌动蛋白。我们将回答三个问题。首先，细菌蛋白如何在入侵过程中促进肌动蛋白的组装？第二，为什么立克次体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，细菌蛋白质如何促进细胞间的传播？更具体地说，在目标1中，我们将测试立克次体是否以及如何使用SCA2、T4SS以及可能的T4SS效应器Sca4、RickA和Ank2来侵袭和毒力，并确定这些因素是否为T4SS效应器。在目标2中，我们将测试早期和晚期运动是否促进通过自噬避免细菌死亡，确定早期和晚期运动之间的转换发生的时间和方式，并研究RickA和sca2介导运动和毒力的分子机制。在目标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在调节不同扩散阶段的肌动蛋白和膜动力学中的重要性。这些研究将揭示宿主-病原体相互作用的新机制，并可能导致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.