Characterizing gene family expansion in an atypical bacterial secretion system

项目摘要 历史记录突出了立克次体疾病的全球影响，致命的节肢动物传播的疾病重新出现 斑疹和斑疹伤寒立克次体病，以及新病原体的出现。专性胞内立克次体 感染节肢动物和脊椎动物的整个生命周期。细胞内的生活方式和还原的富含AT的 立克次体基因组研究对以表征机制为中心的研究提出了巨大的挑战 致病性。然而，研究立克次体的跨学科方法可以克服 这些细菌对传统遗传方法的顽固性。本申请采用这样的方法， 重点研究立克次体VIR同源物(RVH)IV型分泌系统(T4SS)。几乎什么都不知道 RVH机器如何组装以将效应器转移到宿主细胞中。这在很大程度上是由于史无前例的RVH 基因家族的扩张。Rvh含有三个重复的家族(rvhB4，rvhB8，rvhB9)：RvhB4，8，9-I是保守的 相对于其他T4SS中的等价物，而RvhB4，8，9-II进化出了非典型特征。此外，RVH包含 增殖的类VirB6基因(rvhB6a-e)，呈线状排列，含有大的N-端和C-端 分机。值得注意的是，基因家族扩大对RVH结构和功能的意义并没有 之前已经探索过了。在这个提案中，我们将检验这样一个假设，即所有的RVH蛋白都由基因支撑 家庭扩展形成了一个结构独特的T4SS，它协调了复杂的立克次体生命周期(传播 节肢动物媒介和脊椎动物宿主之间)。为了检验我们的假设，我们将定义基因复制在 RVH功能(AIM 1)和RvhB6a的胞外功能(AIM 2)。在目标1下，我们将确定 保守的(RvhB4，8，9-I)和非典型的(RvhB4，8，9-II)副本对RVH的结构和功能有何贡献。这 工作将涉及在不同宿主细胞背景下的基因/蛋白质表达分析的混合，蛋白质-蛋白质 通过筛选和靶向途径的相互作用、RvhB4 ATPase的功能特征和反义 利用RNA技术下调RvhB4、8、9-II的表达。在目标2下，我们将描述VirB6的意义- 通过鉴定已知的细胞外立克次体RvhB6蛋白(RvhB6a)，类似于增殖。这部作品 将涉及不同宿主细胞背景下的基因/蛋白质表达分析、亚细胞定位 RvhB6a结构域的分析、RVH效应器与RvhB6a的共定位以及潜在宿主靶点的识别 RvhB6a胞外区。总而言之，这些研究将使我们能够破译奇怪的RVH 机器在立克次体的特定时间点选择性地将效应器移位到宿主细胞中 细胞内生命周期。重要的是，我们提出的研究对Rickettology来说是新的，并将提供有价值的 除了立克次体致病机理的知识库外，使我们的工作具有很强的创新性。成功后 我们的工作完成后，我们将能够继续进行突变研究，以确定RVH分泌的特征，如 以及了解奇怪的RVH架构的哪些方面适合药物靶向。因此，我们的建议， 虽然具有高度的探索性，但与我们开发治疗方法对抗致命立克次体的最终目标是一致的。

Project Summary The global impact of rickettsial diseases is highlighted by historical records, reemergence of fatal arthropod-borne spotted and typhus fever rickettsioses, and emergence of new pathogens. Obligate intracellular Rickettsia species infect arthropods and vertebrates throughout their lifecycles. The intracellular lifestyle and reductive, AT-rich genomes of Rickettsia spp. pose immense challenges to research centered on characterizing mechanisms of pathogenicity. Nevertheless, a transdisciplinary approach towards studying rickettsiae can overcome the near intractability of these bacteria to conventional genetic methodologies. This application employs such an approach, focusing on the Rickettsiales vir homolog (rvh) type IV secretion system (T4SS). Virtually nothing is known about how the rvh machine assembles to translocate effectors into host cells. This is largely due to unprecedented rvh gene family expansion. rvh contains three duplicate families (rvhB4, rvhB8, rvhB9): RvhB4,8,9-I are conserved relative to equivalents in other T4SSs, while RvhB4,8,9-II have evolved atypical features. Furthermore, rvh contains proliferated VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Remarkably, the significance of gene family expansion in regards to rvh structure and function has not previously been explored. In this proposal we will test the hypothesis that all Rvh proteins underpinned by gene family expansion form a structurally unique T4SS that orchestrates the complex rickettsial lifecycle (transmission between arthropod vector and vertebrate host). To test our hypothesis, we will define the role of gene duplication in rvh function (AIM 1) and characterize the extracellular function of RvhB6a (AIM 2). Under AIM 1, we will determine how conserved (RvhB4,8,9-I) and atypical (RvhB4,8,9-II) duplicates contribute to rvh structure and function. This work will involve a blend of gene/protein expression analyses in various host cell backgrounds, protein-protein interactions via screens and targeted approaches, functional characterization of RvhB4 ATPases, and antisense RNA technology to knock-down of RvhB4, 8, 9-II expression. Under AIM 2, we will describe the significance of VirB6- like proliferation by characterizing the lone Rickettsia RvhB6 protein known to be extracellular (RvhB6a). This work will involve a blend of gene/protein expression analyses in various host cell backgrounds, subcellular localization assays for RvhB6a domains, rvh effector co-localization with RvhB6a, and identification of potential host targets of RvhB6a extracellular domains. Collectively, these studies will allow us to decipher the manner by which the odd rvh machine operates to selectively translocate effectors into host cells throughout specific timepoints of the rickettsial intracellular lifecycle. Importantly, our proposed research is novel to Rickettsiology and will provide a valuable addition to the knowledgebase of rickettsial pathogenesis, making our work highly innovative. Upon successful completion of our work, we will be able to move forward with mutagenesis studies to characterize rvh secretion, as well as understand what aspects of the bizarre rvh architecture are amenable to drug targeting. Thus our proposal, while highly exploratory, is in line with our ultimate goal of developing therapeutics to combat fatal rickettsioses.