Modulation of Host Cell Apoptosis By Pathogenic Rickettsiae

描述（由申请人提供）：属于立克次体的专性细胞内细菌是立克次体疾病的斑点热和斑疹伤寒群（SFG和TG）的病原体，几乎在全世界所有地理位置。落基山斑疹热和流行性斑疹伤寒分别由立克次体和普拉兹克氏体引起，是严重感染，对儿童、老年人和免疫缺陷或误诊/未经治疗的患者可致命。一个主要的临床标志是血管内皮细胞的感染，最终导致血管炎症和损伤。虽然细胞凋亡是一种重要的宿主防御机制，在细菌感染过程中限制增殖和随后的发病机制，但细胞内立克次体的生长/传播需要与目标宿主细胞密切相关。因此，像其他细胞内病原体一样，立克次体也可能在感染早期利用抑制宿主凋亡的策略来维持自身的生存。由我们的实验室鉴定和描述，立克次体内皮细胞感染期间的一种这样的策略是NF-？B，但对斑疹伤寒群生物感染过程中的细胞信号事件和潜在的抗凋亡活性尚不清楚。由于SFG和TG生物的细胞内行为存在显著差异，并且有可能故意使用普拉兹氏弧菌（CDC清单上的B类）和立克次体（C类）作为生物恐怖剂，因此解决立克次体发病机制现有知识中的这一关键空白是非常重要的。本探索性资助申请的目的是研究立克次体在感染早期触发宿主细胞抗凋亡机制的假设，以促进感染的建立和进展，并通过同时进行全面的比较分析，确定SFG和TG立克次体利用的宿主信号机制的潜在相似性或差异性。Aim 1旨在鉴定代表性立克次体物种诱导的凋亡和抗凋亡信号通路，并将重点关注促凋亡和抗凋亡基因与Bcl-2、IAP和p53家族蛋白之间平衡的“转移”。Aim 2将进一步破译立克次体感染过程中细胞凋亡调控的复杂机制，通过识别外源性或内在级联反应的参与，以及识别上游的启动物和下游的效应caspases。采用常规生化和最先进的显微/分子遗传学方法相结合，我们将揭示立克次体介导的细胞凋亡调节的分子基础的新见解，并提高我们对寄生细胞内病原体操纵宿主功能的独特视角的理解。获得宿主细胞信号适应性的新知识以及立克次体生物基因组和蛋白质组学表征的最新进展将使我们能够识别和分离潜在的立克次体抗凋亡活性（ies），这可以有针对性地开发新的和改进的策略，旨在提高早期清除感染细胞的能力，以避免播散性内皮感染和相关的血管病变。

DESCRIPTION (provided by applicant): The obligate intracellular bacteria belonging to Rickettsia species are etiological agents of the spotted fever and typhus groups (SFG and TG) of rickettsial diseases in almost all geographic locations worldwide. Rocky Mountain spotted fever and epidemic typhus, caused respectively by R. rickettsii and R. prowazekii, are serious infections that can be fatal in children, elderly, and immunodeficient or misdiagnosed/untreated patients. A major clinical hallmark is the infection of endothelial cell lining of vessels, ultimately leading to vascular inflammation and damage. While apoptosis serves as an important host defense mechanism to restrict proliferation and ensuing pathogenesis during bacterial infections, intracellular rickettsiae require intimate association with the target host cells for their growth/spread. It is thus possible that like other intracellular pathogens, rickettsiae may also utilize strategies to inhibit host apoptosis early during the infection for sake of their own survival. Identified and described by our laboratory, one such strategy during endothelial cell infection of R. rickettsii is the activation of NF-?B, but nothing is known about cell signaling events during infection with and potential anti-apoptotic activities of typhus group organisms. To address this critical gap in the existing knowledge of rickettsial pathogenetic mechanisms is highly significant because of distinctive differences in the intracellular behavior of SFG and TG organisms and the potential for intentional use of R. prowazekii (Class B on CDC list) and R. rickettsii (Category C) as bioterror agents. The objectives of this exploratory grant application are to investigate the hypothesis that early during the infection, rickettsiae trigger anti-apoptotic mechanism(s) in host cells to facilitate the establishment and progression of infection and to define potential similarities or differences in host signaling mechanisms exploited by SFG and TG rickettsiae by conducting simultaneous and thorough comparative analyses. Aim 1 is designed to identify apoptotic and anti-apoptotic signaling pathways induced by representative Rickettsia species and will focus on 'shift(s)' in the equilibrium between pro- and anti-apoptotic genes and proteins of Bcl-2, IAP, and p53 families. Aim 2 will further decipher the complex mechanistic aspects of apoptosis regulation during Rickettsia infection by discerning the involvement of extrinsic or intrinsic cascades and identifying the upstream initiator and downstream effector caspases. Employing a combination of conventional biochemical and state of the art microscopic/molecular genetic approaches, we will reveal novel insights into the molecular basis of Rickettsiae-mediated modulation of apoptosis and improve our understanding of the unique perspective of manipulation of host functions by parasitic intracellular pathogens. Acquisition of this new knowledge regarding adaptations in host cell signaling and recent progress in the genomic and proteomic characterization of Rickettsia organisms will enable us to identity and isolate potential rickettsial anti-apoptotic activity(ies), which can be targeted to develop new and improved strategies aimed at enhancing clearance of infected cells early to avoid disseminated endothelial infection and associated vascular pathologies.