Murine Typhus

节肢动物传播的立克次体是人类最致命的病原体之一 并引起全球公共卫生关注。我们在过去三年半的工作中， 对R.伤寒的发病机制，特别是， 立克次体的分泌蛋白。在这份提案中，我们将讨论基本问题， 立克次体病原体生物学中两个未充分研究但非常重要的主题中的问题。第一、 我们将确定R.猫蚤（猫栉首蚤） 先天免疫，微生物群和其他因素，以建立感染和促进传播， 脊椎动物宿主;和第二，定义功能谱和免疫原性潜力的脊椎动物宿主。 R.伤寒杆菌分泌磷脂酶的军械库，改变宿主膜生理学， 吞噬体逃逸和细胞内复制。我们将实施跨学科的方法 (e.g.,基因组学、细菌学、昆虫学和细胞/分子生物学）， 调查这些重点领域，如以下具体目标所述。在Aim I中，我们将 破译调节因子R。伤寒杆菌在C.猫和促进传播 通过1）稳健的组织特异性时程表达分析，2）沉默IMD和Toll 途径基因表达; 3）检测分离的R.伤寒 肽聚糖，以及4）表征C.微生物组对感染的影响在Aim II中， 我们将描述R.伤寒分泌型磷脂酶我们将 用两种方法刻画R。伤寒磷脂酶：1）Pat 1的功能表征， Pat 2和Pld通过测定它们的亚细胞定位、脂质识别和内相互作用组来研究。 宿主细胞，下游分析旨在揭示这些相互作用的意义， 介导细菌进入宿主胞质溶胶;和; 2）炎性体组分的鉴定 是R.伤寒，并确定Pat 1，Pat 2和Pld活动的影响 炎性小体激活这些研究将大大促进我们对 复杂的立克次体/载体/宿主关系，揭示了立克次体寄生虫如何 在专性立克次体的整个生命周期中，节肢动物向脊椎动物病原体转变。 因此，我们预计我们的工作将使我们更接近于产生更谨慎的治疗方法， 对抗致命的立克次体病。

Arthropod-borne Rickettsia species are among the most virulent and lethal human pathogens and are of significant global Public Health concern. Our work over the past 3.5 years resulted in significant progress in understanding of R. typhi pathogenesis, and in particular, the role of the rickettsial secretome during host cell invasion. For this proposal, we will address fundamental questions in two understudied yet highly significant topics in rickettsial pathogen biology. First, we will determine the mechanisms by which R. typhi modulates cat flea (Ctenocephalides felis) innate immunity, microbiota and other factors to establish infection and facilitate transmission to the vertebrate host; and second, defining the functional spectrum and immunogenic potential of the R. typhi armory of secreted phospholipases that alter host membrane physiology to facilitate phagosomal escape and intracellular replication. We will implement a transdisciplinary approach (e.g., phylogenomics, bacteriology, entomology, and cellular/molecular biology) to thoroughly investigate these focal areas, as outlined by the following Specific Aims. In Aim I, we will decipher the modulatory factors R. typhi employs to colonize C. felis and facilitate transmission via 1) a robust tissue-specific, time-course expression analysis, 2) silencing IMD and Toll pathway gene expression, 3) testing the immunomodulatory propensities of isolated R. typhi peptidoglycan, and 4) characterizing the impact of the C. felis microbiome on infection. In Aim II, we will characterize the function and immunogenicity of R. typhi secretory phospholipases. We will take two approaches to characterize R. typhi phospholipases: 1) functional characterization of Pat1, Pat2 and Pld via determining their subcellular localization, lipid recognition and interactome within host cells, with downstream analyses designed to reveal the significance of these interactions in mediating bacterial access to the host cytosol; and; 2) identification of inflammasome components necessary for IL-1β secretion by R. typhi, and determining the impact of Pat1, Pat2, and Pld activities on inflammasome activation. Together these studies will significantly advance our understanding of the complex Rickettsia/vector/host relationship, shedding light on how rickettsial parasites of arthropods transition to vertebrate pathogens throughout the obligatory Rickettsia life cycle. Thus, we anticipate that our work will take us closer to generating more prudent therapeutics to combat fatal rickettsioses.