Modulation of Host Cell Apoptosis By Pathogenic Rickettsiae

描述(申请人提供)：属于立克次体的专性胞内细菌是世界上几乎所有地理位置的立克次体疾病斑点热群和斑疹伤寒群(SFG和TG)的病原体。落基山斑点热和流行性斑疹伤寒分别由立克次氏杆菌和普罗瓦泽克杆菌引起，是严重的感染，对儿童、老年人和免疫缺陷或误诊/未治疗的患者可能是致命的。一个主要的临床特征是血管内皮细胞的感染，最终导致血管炎症和损伤。在细菌感染过程中，细胞凋亡是一种重要的宿主防御机制，以限制其增殖和随后的致病过程，而细胞内的立克次体则需要与目标宿主细胞密切结合才能生长/传播。因此，与其他细胞内病原体一样，立克次体也可能为了自身的生存而在感染早期利用抑制宿主细胞凋亡的策略。我们实验室鉴定和描述了里氏杆菌内皮细胞感染过程中的一种这样的策略是激活核因子-β，但对斑疹伤寒杆菌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。解决现有立克次体致病机制知识中的这一关键差距具有非常重要的意义，因为SFG和TG生物在细胞内行为的显著差异，以及故意将普氏立克次体(CDC名单上的B类)和立克次体(C类)用作生物恐怖制剂的可能性。这项探索性拨款申请的目的是调查这一假说，即在感染早期，立克次体在宿主细胞中触发抗凋亡机制(S)，以促进感染的建立和进展，并通过进行同步和彻底的比较分析，确定SFG和TG立克次体利用的宿主信号机制的潜在相似或不同之处。目的1旨在鉴定典型立克次体诱导的凋亡和抗凋亡信号通路，并将重点放在促进和抗凋亡基因和蛋白在Bcl2、IAP和P53家族之间平衡的‘转变(S)’。目的2通过辨别外在或内在的级联反应，识别上游启动子和下游效应子caspase，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。结合传统的生化方法和最新的显微/分子遗传学方法，我们将揭示立克次体介导的细胞凋亡调控的分子基础，并加深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。获得这种关于宿主细胞信号适应的新知识和最近在立克次体基因组和蛋白质组特征方面的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，这可以被靶向地开发新的和改进的策略，旨在加强感染细胞的早期清除，以避免播散性内皮感染和相关的血管病理。

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.