Murine Typhus

节肢动物传播的立克次体是最致命的人类病原体之一。 并对全球公共卫生造成重大影响。我们在过去3.5年的工作取得了成果 在理解伤寒杆菌致病机制方面取得了重大进展，尤其是伤寒杆菌 寄主细胞入侵时的立克次体分泌体。对于这项提案，我们将从根本上解决 立克次体病原体生物学中两个未被充分研究但意义重大的课题中的问题。第一, 我们将确定伤寒杆菌调节猫蚤(Ctenocephalides Felis)的机制。 先天免疫、微生物区系和其他因素，以确定感染和促进传播到 第二，定义了脊椎动物宿主的功能谱和免疫原性 伤寒沙门氏菌的分泌型磷脂酶，改变宿主膜的生理，以促进 吞噬小体逃逸和细胞内复制。我们将实施跨学科的方法 (例如，系统基因组学、细菌学、昆虫学和细胞/分子生物学) 调查这些重点领域，具体目标如下。在Aim I中，我们将 破译伤寒沙门氏菌定植和促进传播的调节因子 通过1)强大的组织特异性、时程表达分析，2)抑制IMD和Toll 途径基因表达，3)检测伤寒杆菌分离株的免疫调节倾向 以及4)确定猫毛虫微生物群对感染的影响。在AIM II中， 我们将对伤寒杆菌分泌型磷脂酶的功能和免疫原性进行鉴定。我们会 采用两种方法鉴定伤寒沙门氏菌磷脂酶：1)Pat1的功能鉴定， Pat2和PLD通过测定它们在体内的亚细胞定位、脂质识别和相互作用 宿主细胞，下游分析旨在揭示这些相互作用在 介导细菌进入宿主胞浆；以及；2)炎性小体成分的鉴定 伤寒沙门氏菌分泌IL-1β所必需的，以及PAT1、PAT2和PLD活性的影响 关于炎症体的激活。总之，这些研究将极大地促进我们对 立克次体/媒介/宿主的复杂关系，揭示了立克次体寄生虫是如何 节肢动物在整个强制性立克次体生命周期中向脊椎动物病原体过渡。 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 与致命的立克次体病作斗争。

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.