Role of mTOR signaling in endothelial responses to Rickettsia rickettsii infection.

项目摘要/摘要 立克次体病作为传染病，因其对全球人类的历史性和持续性影响而脱颖而出 健康。革兰氏阴性革兰氏阴性立克次体(RR)引起的落基山斑点热 胞内α-变形杆菌，是美国最严重的立克次体之一，也是一种应报告的疾病。一个 人类疾病发病机制和已建立的感染实验室模型的重要决定因素是 血管微血管内皮细胞播散性感染，导致立克次体血管炎 定义为血管系统炎症和导致组织液的血管通透性受损 重要器官失衡和浮肿。RR诱导的内皮反应包括获得前黏附分子， 促凝剂、促炎性表型和主要转录调节因子核因子的激活- κB，然而决定先天免疫反应和宿主细胞命运的潜在机制代表了一种 被忽视的基本相关领域。值得注意的是，自噬作为“双刃剑”宿主的潜在作用 防御机制能够支持细胞内病原体，抗炎内皮细胞反应 最大限度地减少血管损伤和功能障碍，以及促进与抗炎信号的平衡 立克次体毒力的生物学基础代表着科学研究的未知领域。我们拿出证据 提示信号转导和转录激活蛋白STAT3的磷酸化增加 反应与自噬和雷帕霉素机械靶点(MTOR)的激活相一致 RR感染人内皮细胞过程中的复合物C_1和C_2我们耐人寻味的初步发现和 大量发表的关于RR诱导转录激活的工作是我们假设的基础 MTOR是促炎/促黏附平衡的核心调节决定因素 RR感染血管内皮细胞的抗炎天然免疫反应。考虑到mTOR信令 涉及两个结构和功能不同的复合体，目标1将解决根本上重要的 RR株内皮细胞感染过程中mTORC1和mTORC2激活状态的确定 并确定它们对宿主细胞自噬和立克次体复制的抑制作用。 然后，Aim 2将破译mTORC1和C2在感染诱导的炎症中可能的不同作用 发信号。我们将战略性地采用当代、前沿和跨学科的CELL和 分子生物学将获得对宿主mTORC1/C2激活的新的和独特的见解 立克次体毒力及其在调节自噬和亲体免疫应答中的作用 /使用体外和体内感染模型的抗炎信号。关于病原体分析的建议 与能够调节有益和/或有害宿主响应的新型信号检查点的相互作用 将指导创新的治疗策略，以对抗与(重新)出现的立克次体相关的后遗症。

PROJECT SUMMARY/ABSTRACT Rickettsioses as infectious diseases stand out for their historic and continued impact on the global human health. Rocky Mountain spotted fever due to tick-transmitted Rickettsia rickettsii (Rr), a Gram-negative obligate intracellular α-proteobacterium, is one of the most severe rickettsioses and a notifiable illness in the USA. An important determinant of disease pathogenesis in humans and established laboratory models of infection is disseminated infection of microvascular endothelial cell lining of blood vessels, resulting in ‘rickettsial vasculitis’ defined by inflammation of the vasculature and compromised vascular permeability leading to tissue fluid imbalance and edema of vital organs. Rr-induced endothelial responses include acquisition of pro-adhesive, pro-coagulant, and pro-inflammatory phenotypes and activation of master transcription regulator nuclear factor- κB, yet the mechanisms underlying determination of innate immune responses and host cell fate represent a neglected area of fundamental relevance. Notably, potential roles of autophagy as a ‘double-edged’ host defense mechanism capable of supporting intracellular pathogens, anti-inflammatory endothelial responses to minimize vascular damage and dysfunction, and the balance of pro- vis-à-vis anti-inflammatory signaling as the biological basis of rickettsial virulence represent unexplored arenas of scientific enquiry. We present evidence suggesting increased phosphorylation of signal transducer and activator of transcription protein STAT3, a response coinciding with the onset of autophagy and activation of mechanistic target of rapamycin (mTOR) complexes C1 and C2 during Rr infection of human endothelial cells. Our intriguing preliminary findings and extensive published work on Rr-induced transcriptional activation serve as the basis of our hypothesis that mTOR functions as a central regulatory determinant of the balance of pro-inflammatory/pro-adhesive vis-a-vis anti-inflammatory innate immune responses of Rr-infected endothelium. Considering that mTOR signaling involves two structurally and functionally distinct complexes, Aim 1 will address the fundamentally important definition of the activation status of mTORC1 and mTORC2 during endothelial infection with Rr strains of varying virulence and determine the effects of their inhibition on host cell autophagy and rickettsial replication. Aim 2 will then decipher potentially differential roles of mTORC1 and C2 in infection-induced inflammatory signaling. We will strategically employ contemporary, cutting-edge, and interdisciplinary approaches of cell and molecular biology to gain new and unique insights into host mTORC1/C2 activation in correlation with rickettsial virulence and their roles in governing innate immune responses via regulation of autophagy and pro- /anti-inflammatory signaling using in vitro and in vivo models of infection. The proposed analysis of pathogen interactions with novel signaling checkpoints capable of regulating beneficial and/or detrimental host responses will guide innovative therapeutic strategies to combat the sequelae associated with (re)emerging rickettsioses.