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

立克次体侵袭、细胞内存活和基于肌动蛋白的运动的机制

• 批准号: 10238082
• 负责人: Matthew D Welch
• 金额: $ 38.01万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238082
• 关键词: Actins; Address; Automobile Driving; Autophagocytosis; Bacteria; Bacterial Physiology; Cell Surface Receptors; Cells; Cellular Structures; Cellular biology; Cytoskeleton; Cytosol; Diagnosis; Diagnostic; Disease; Endothelium; Funding; Genes; Genome; Growth; Human; In Vitro; Infection; Invaded; Knowledge; Lead; Mediating; Membrane; Membrane Proteins; Molecular; Mus; Mutagenesis; Mutation; Nutrient; Pathogenesis; Phagosomes; Phase; Phenotype; Phospholipase; Play; Polymers; Process; Proteins; Publishing; Rickettsia; Rickettsia Infections; Rickettsia parkeri; Role; Study models; Surface Antigens; Testing; Typhus; Virulence; Work; bacterial genetics; base; cell motility; human disease; improved; in vivo; innovation; macrophage; mouse model; mutant; novel strategies; pathogen; pathogenic bacteria; polymerization; receptor; spotted fever; ubiquitin ligase; unpublished works

PROJECT SUMMARY/ABSTRACT The Rickettsiae are obligate intracellular bacterial pathogens that cause serious diseases, such as spotted fever and typhus. We study the model spotted fever group (SFG) species Rickettsia parkeri, which causes an eschar-associated human rickettsiosis, is experimentally tractable, and has emerging mouse models of pathogenesis, making it ideal for revealing molecular mechanisms of SFG Rickettsia infection and virulence. During infection, SFG Rickettsia invade host cells by mobilizing the actin cytoskeleton, escape from the phagosome into the cytosol, replicate while avoiding degradation by autophagy, and harness actin polymerization 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 components to promote their infection cycle. To bridge these gaps, in the current funding period we have pioneered an innovative combination of bacterial genetics and host cell biology to identify key Rickettsia factors that manipulate host cells. In unpublished work, we discovered that outer membrane protein OmpB is crucial for both invasion and avoidance of autophagy. We also observed that patatin-like phospholipase Pat1 plays a role in phagosome escape and/or autophagy evasion. Additionally, in published work, we demonstrated that Rickettsia use two actin-polymerizing surface proteins to direct sequential phases of motility – with RickA driving early motility and surface cell antigen Sca2 driving late motility. However, key outstanding questions remain, including: How do Rickettsia engage host receptors to promote invasion? How do Rickettsia degrade membranes during phagosome escape or inhibit membrane engulfment to avoid autophagy? And how do Rickettsia coordinate and use two actin assembly factors in distinct phases of motility? Our preliminary and published findings suggest the overall hypothesis that OmpB, Pat1, RickA, and Sca2 are multifunctional proteins that mobilize or disrupt host cell components and play a crucial role in infection in vivo. This hypothesis will be tested in three Aims focused on uncovering the mechanisms through which OmpB, Pat1, RickA, and Sca2 influence invasion, intracellular survival, and motility. The Aims are to: (1) define the role of Rickettsia surface protein OmpB in invasion and intracellular survival; (2) investigate the role of Pat1 phospholipase in phagosome escape and intracellular survival; and (3) determine how and why Rickettsia use two distinct actin-based motility mechanisms. The proposed studies will advance the field by revealing 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.

项目总结/摘要 立克次氏体是专性细胞内细菌病原体，其引起严重疾病，如斑点 发烧和斑疹伤寒我们研究了斑点热组（SFG）的模式物种帕克氏立克次体，它引起了一个 焦痂相关的人类立克次体病，是实验上易于处理的，并有新兴的小鼠模型， 致病机制，使其成为揭示SFG立克次体感染和毒力的分子机制的理想方法。 在感染过程中，SFG立克次体通过动员肌动蛋白细胞骨架侵入宿主细胞，逃离宿主细胞， 吞噬体进入胞质溶胶，复制，同时避免自噬降解，并利用肌动蛋白 聚合以促进细胞内运动和细胞-细胞扩散。然而，在以下方面存在根本性差距： 我们对SFG立克次体利用或破坏宿主细胞成分的分子机制的了解 来促进它们的感染循环为了弥补这些差距，在目前的供资期内，我们开创了一个 细菌遗传学和宿主细胞生物学的创新组合，以确定关键的立克次体因子， 操纵宿主细胞在未发表的工作中，我们发现外膜蛋白OmpB对于 入侵和避免自噬。我们还观察到，patatin样磷脂酶Pat 1在 吞噬体逃逸和/或自噬逃避。此外，在已发表的工作中，我们证明， 立克次体使用两种肌动蛋白聚合表面蛋白来指导运动的连续阶段-RickA 驱动早期运动和表面细胞抗原Sca 2驱动晚期运动。然而，关键的未决问题 保留，包括：立克次体如何从事宿主受体，以促进入侵？立克次体如何降解 在吞噬体逃逸或抑制膜吞噬，以避免自噬？怎么 立克次体协调和使用两个肌动蛋白组装因子在不同阶段的运动？我们的初步和 已发表的研究结果表明，OmpB、Pat 1、RickA和Sca 2是多功能的 动员或破坏宿主细胞成分并在体内感染中起关键作用的蛋白质。这 假设将在三个目标中进行测试，重点是揭示OmpB，Pat 1， RickA和Sca 2影响侵袭、细胞内存活和运动性。其目的是：（1）确定 立克次体表面蛋白OmpB在侵袭和细胞内存活中的作用;（2）探讨Pat 1在立克次体侵袭和细胞内存活中的作用 磷脂酶在吞噬体逃逸和细胞内存活中的作用;（3）确定立克次体如何和为什么使用磷脂酶 两种不同的肌动蛋白运动机制拟议中的研究将通过揭示关键的 立克次体和其他病原体操纵宿主细胞的分子机制以及 这些传染性的机制。我们的研究也可能导致改善诊断和治疗， 立克次体和其他感染。