ACTIN BASED MOTILITY BY RICKETTSIA RICKETTSII

描述(改编自申请者摘要)：斑点热组 立克次体是人类严重疾病的病原体 包括由立克次体引起的落基山斑点热病。 由于缺乏可行的遗传系统和 使用真核细胞作为生长介质的必要性，有一个 缺乏关于这些病毒所利用的特定毒力机制的信息 有机体。对SFG立克次体毒力的洞察最近得到了 通过发现，当进入细胞质隔间时， 生物体招募和聚合宿主细胞肌动蛋白以促进直接 细胞间的传播。为了更好地了解立克次体 基于肌动蛋白的运动性，这项提案中进行的研究具有 以下目标：1)确定立克次体蛋白配体(S) 介导基于肌动蛋白的运动和结合的同源胞浆蛋白 该配体(S)，和2)完全表征立克次体肌动蛋白尾部结构 和队形。与肌动蛋白有关的立克次体蛋白 招募很可能是表面上的本地化。因此，目标1将重点放在 两种免疫显性立克次体外膜蛋白的潜在参与 膜蛋白、Rompa和rOmpB。转染法生产Vero细胞 Rompa或rOmpB的特定结构域，并感染立克次体，将被 检查以确定胞质ROMP结构域的表达是否中断 立克次体肌动蛋白募集和/或宿主细胞微丝结构。一个 并行方法将利用Rompa或rOmpB特异性单抗的显微注射 以确定立克次体是否诱导了 肌动蛋白聚合可以被抑制。因为立克次体表面 可能涉及Rompa和rOmpB以外的蛋白质，全球筛查 紫外线照射产生的立克次体小斑块形成突变株将被 在目标2中进行，以确定缺乏肌动蛋白的立克次体突变体 动员。突变和野生型立克次体的蛋白质谱 进行比较，以确定并允许对候选人进行分子克隆 立克次体肌动蛋白募集蛋白。目标3将识别和克隆主机 与已鉴定的立克次体肌动蛋白相互作用的胞浆蛋白 招募蛋白。相互作用的宿主蛋白将被初步鉴定 作为与产生的立克次体蛋白免疫共沉淀的蛋白质 通过转染法在Vero细胞中表达。将进行亲和层析 用细胞质提取物来纯化相互作用的宿主蛋白，并允许 编码基因的鉴定和克隆。一项单独的战略将 采用酵母双杂交技术。最后，目标4将检查立克次体 肌动蛋白尾巴识别肌动蛋白细丝长度和方向，相关 宿主肌动蛋白结合蛋白和立克次体的相对速率 基于肌动蛋白的运动。这些研究不仅将导致更大的 对立克次体致病分子机制的了解，但他们 也将提供对肌动蛋白聚合的动态控制的洞察。

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.