ACTIN BASED MOTILITY BY RICKETTSIA RICKETTSII

立克次体 RICKETTSII 基于肌动蛋白的运动

• 批准号: 2680507
• 负责人: Robert A. Heinzen
• 金额: $ 11.23万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-15 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2680507
• 关键词: Rickettsia; Rocky Mountain spotted fever; Vero cells; actins; affinity chromatography; bacterial proteins; cell motility; host organism interaction; intermolecular interaction; ligands; membrane proteins; microinjections; molecular cloning; monoclonal antibody; protein structure function; transfection; yeast two hybrid system

DESCRIPTION (Adapted from the applicant's abstract): Spotted fever group (SFG) rickettsiae are the etiologic agents of serious human diseases including Rocky Mountain spotted fever, caused by Rickettsia rickettsii. Because of limitations imposed by the lack of workable genetic systems and the necessity of using eucaryotic cells as a growth medium, there is a paucity of information on specific virulence mechanisms exploited by these organisms. Insight into the virulence of SFG rickettsiae has recently been achieved with the discovery that, upon entering the cytoplasmic compartment, organisms recruit and polymerize host cell actin to promote direct cell-to-cell spread. To achieve a greater understanding of rickettsial actin-based motility, the research conducted in this proposal has the following goals: 1) identify the rickettsial protein ligand(s) that mediates actin-based motility and the cognate cytosolic proteins that bind this ligand(s), and 2) fully characterize rickettsial actin tail structure and formation. A priori, the rickettsial protein involved in actin recruitment is likely surface localized. Therefore, aim 1 will focus upon the potential involvement of the two immunodominant rickettsial outer membrane proteins, rOmpA and rOmpB. Transfected Vero cells producing specific domains of rOmpA or rOmpB, and infected with R. rickettsii, will be examined to determine whether cytosolic rOmp domain expression disrupts rickettsial actin recruitment and/or host cell microfilament structure. A parallel approach will utilize microinjection of rOmpA or rOmpB-specific Mab into the cytoplasm of infected cells to determine if rickettsial-induced actin polymerization can be inhibited. Because a rickettsial surface protein other than rOmpA and rOmpB may be involved, a global screen of rickettsial small-plaque-forming mutants generated by UV irradiation will be conducted in aim 2 to identify rickettsial mutants deficient for actin mobilization. The protein profiles of mutant and wild type rickettsiae will be compared to identify and allow molecular cloning of the candidate rickettsial actin recruitment protein. Aim 3 will identify and clone host cytosolic proteins that interact with the identified rickettsial actin recruitment protein. Interacting host proteins will initially be identified as proteins that co-immunoprecipitate with the rickettsial protein produced in Vero cells by transfection. Affinity chromatography will be performed with cytoplasmic extracts to purify the interacting host protein and allow identification and cloning of the encoding gene. A separate strategy will employ yeast-two hybrid technology. Finally, aim 4 will examine rickettsial actin tails to identify actin filament length and orientation, associated host actin-binding proteins, and the relative rate of rickettsial actin-based movement. Not only will these studies lead to a greater understanding of molecular mechanisms of rickettsial pathogenesis, but they will also provide insight into the dynamic control of actin polymerization.

描述（改编自申请人摘要）：斑点热组 (SFG)立克次氏体是严重人类疾病的病原体 包括由立克次体引起的落基山斑疹热。 由于缺乏可行的遗传系统所带来的限制， 使用真核细胞作为生长培养基的必要性， 缺乏关于这些病毒利用的特定毒力机制的信息， 有机体 最近，对SFG立克次体毒力的深入了解， 这一发现的实现是，一旦进入细胞质区室， 生物体募集并激活宿主细胞肌动蛋白， 细胞间传播 为了更好地了解立克次体 肌动蛋白为基础的运动，在这项建议进行的研究有 以下目标：1）鉴定立克次体蛋白配体， 介导肌动蛋白为基础的运动和同源的胞质蛋白， 该配体，和2）充分表征立克次体肌动蛋白尾部结构 和形成。 先验地，与肌动蛋白有关的立克次体蛋白 募集可能是表面局部的。 因此，目标1将侧重于 两种免疫显性立克次体外膜 膜蛋白rOmpA和rOmpB。 转染的Vero细胞产生 rOmpA或rOmpB的特异性结构域，并感染R.立克次氏体，将是 检查以确定胞质rOmp结构域表达是否破坏 立克次体肌动蛋白募集和/或宿主细胞微丝结构。 一 平行方法将利用rOmpA或rOmpB特异性Mab的显微注射 感染细胞的细胞质中，以确定立克次体诱导的 可以抑制肌动蛋白聚合。 因为立克次体表面 除了rOmpA和rOmpB以外的蛋白质可能参与， 通过紫外线照射产生的立克次氏体小空斑形成突变体将被 在目标2中进行，以鉴定肌动蛋白缺陷的立克次体突变体 动员。 突变体和野生型立克次体的蛋白质谱将 进行比较，以确定并允许候选物的分子克隆 立克次体肌动蛋白募集蛋白。 目标3将识别和克隆主机 与鉴定的立克次体肌动蛋白相互作用的胞浆蛋白 募集蛋白 相互作用的宿主蛋白质将首先被鉴定 作为与产生的立克次体蛋白共免疫沉淀的蛋白质 在Vero细胞中进行转染。 将进行亲和层析 用细胞质提取物纯化相互作用的宿主蛋白， 编码基因的鉴定和克隆。 另一项战略将 采用酵母双杂交技术。 最后，aim4将检查立克次体 肌动蛋白尾，以确定肌动蛋白丝的长度和方向，相关 宿主肌动蛋白结合蛋白和立克次体的相对比率 肌动蛋白为基础的运动 这些研究不仅会导致更大的 了解立克次体发病机制的分子机制，但他们 也将提供深入了解动态控制肌动蛋白聚合。