Mechanisms of Rickettsia invasion, intracellular survival, and actin-based motility

项目总结/摘要 致病性立克次体是专性细胞内细菌，可引起斑疹热和斑疹伤寒等疾病。 我们研究斑点热组（SFG）物种立克次体parkeri，这导致焦痂相关的人类 立克次体病，实验上易于处理，使其成为揭示立克次体病分子机制的理想模型。 立克次体感染和毒力。在侵入宿主细胞后，SFG立克次体逃离宿主细胞。 吞噬体进入胞质溶胶，复制，同时避免泛素化和自噬，并将宿主肌动蛋白 促进细胞内运动和细胞-细胞扩散。然而，在我们的知识中存在着根本性的空白。 SFG立克次体利用或破坏宿主细胞结构和途径促进 感染周期。为了弥合这些差距，在目前的赠款期间，我们发现，patatin- 与磷脂酶Pat 1一样，Pat 1对毒力和从宿主膜逃逸也很重要， 吞噬体和自噬体。我们进一步表明，外膜蛋白OmpB和赖氨酸 甲基化对于毒性和保护细菌免于泛素化和自噬是至关重要的。我们也 观察到两种基于肌动蛋白的运动蛋白RickA和Sca 2在细胞-细胞扩散中独立发挥作用， 毒性。最后，我们建立了焦痂相关立克次体病的干扰素受体缺陷小鼠模型 可用于评估细菌因子在毒力中的作用。这些发现支持了整体科学 立克次体蛋白操纵宿主细胞成分使细菌从宿主逃逸的前提 膜，避免宿主泛素化途径，以及动员宿主细胞骨架进行运动。 然而，关键的未决问题依然存在。细菌磷脂酶活性如何促进感染？ 细菌表面结构如何阻止宿主机器的泛素化？立克次体如何以及为何 使用两种机制来进行细胞内运动？我们将解决这些悬而未决的问题，测试 细菌分泌蛋白和表面蛋白的结构、功能和调节至关重要的总体假设 操纵或避免宿主细胞分子和结构，使细胞感染和毒力， 动物这个一般假设将在三个目标中进行测试，重点是揭示Pat 1，OmpB， RickA和Sca 2在逃离宿主膜、细胞内存活和运动中的作用。目标是：（1） 确定立克次体磷脂酶活性如何促进感染;（2）揭示立克次体表面 结构能够避免泛素化;（3）确定立克次体如何以及为什么使用两种机制 细胞内的运动。其影响将是揭示立克次体使用的关键分子机制， 其他病原体操纵宿主细胞和这些机制对感染性的重要性。我们的研究 还可能导致立克次体和其他感染的诊断和治疗的改进。

PROJECT SUMMARY/ABSTRACT Pathogenic Rickettsiae are obligate intracellular bacteria that cause diseases such as spotted fever and typhus. We study the spotted fever group (SFG) species Rickettsia parkeri, which causes an eschar-associated human rickettsiosis and is experimentally tractable, making it an ideal model for revealing molecular mechanisms of SFG Rickettsia infection and virulence. Following invasion of host cells, SFG Rickettsia escape from the phagosome into the cytosol, replicate while avoiding ubiquitylation and autophagy, and polymerize host actin to promote intracellular motility and cell-cell spread. However, there are fundamental gaps in our knowledge of the molecular mechanisms by which SFG Rickettsia exploit or disrupt host cell structures and pathways to promote their infection cycle. Towards bridging these gaps, in the current granting period we discovered that the patatin- like phospholipase enzyme Pat1 is important for virulence and for escaping from host membranes including phagosomes and autophagosomes. We further showed that outer membrane protein OmpB and lysine methylation are crucial for virulence and for shielding bacteria from ubiquitylation and autophagy. We also observed that two actin-based motility proteins, RickA and Sca2, function independently in cell-cell spread and virulence. Finally, we developed an interferon receptor-deficient mouse model of eschar-associated rickettsiosis that can be used to evaluate the role of bacterial factors in virulence. These findings support the overall scientific premise that rickettsial proteins manipulate host cell components to enable bacterial escape from host membranes, avoidance of host ubiquitylation pathways, and mobilization of the host cytoskeleton for movement. However, key outstanding questions remain. How does bacterial phospholipase activity contribute to infection? How does bacterial surface architecture prevent ubiquitylation by host machinery? How and why do Rickettsia use two mechanisms for intracellular movement? We will address these outstanding questions, testing the overall hypothesis that the structure, function, and regulation of bacterial secreted and surface proteins is critical for manipulating or avoiding host cell molecules and structures, enabling infection of cells and virulence in animals. This general hypothesis will be tested in three Aims focused on uncovering the roles of Pat1, OmpB, RickA, and Sca2 in escape from host membranes, intracellular survival, and motility. The Aims are to: (1) determine how Rickettsia phospholipase activity contributes to infection; (2) reveal how Rickettsia surface architecture enables avoidance of ubiquitylation; and (3) establish how and why Rickettsia use two mechanisms for intracellular movement. The impact will be to reveal crucial molecular mechanisms used by Rickettsia and other pathogens to manipulate host cells and the importance of these mechanisms to infectivity. Our studies may also lead to improved diagnostics and treatments for rickettsial and other infections.