Investigating genes of unknown function required for Rickettsia parkeri infection

项目摘要/摘要 细胞内的细菌病原体通过大量的机制操纵宿主细胞。研究这些 相互作用推动了我们对针对这些病原体和宿主细胞的治疗发展的理解 生物学。然而，许多细胞内的病原体由于它们的专性和 对基因操纵的抵抗。这些包括斑点热群(SFG)立克次体，它会引起一系列 潜在严重的节肢动物传播的人类疾病，包括落基山斑疹热。这个 SFG立克次体新的随机诱变系统的发展推动了对这些微生物的研究 并暗示了该属史无前例的病原体创新的显著多样性。最近，我们的实验室 在模式立克次体物种帕氏立克次体中进行小规模转座子突变筛选以鉴定 弱化的突变体。这一筛选导致分离出具有感染缺陷的&gt；100个帕克氏杆菌突变体，只有 少数含有先前与帕克氏杆菌毒力相关的基因的插入。其余的菌株代表一种 是探索和了解帕氏杆菌和细胞内病原体生物学的宝贵工具。超过15%的基因 此屏幕中的点击未加注释。其中两个未被注释的基因hrtA和sp50编码帕氏杆菌蛋白。 它们被预测为暴露在表面或分泌出来，并具有推测的结构特征，提示直接 与宿主蛋白结合。我推测HrtA和Sp50是新的帕氏杆菌分泌或表面暴露的 可以劫持特定宿主功能以促进感染的效应器。在这项提案中，我将首先演示 HrtA和Sp50的时空生态位(目标1)，以确定它们如何对帕克氏杆菌做出表型贡献 感染。然后，我将使用亲和纯化方法来鉴定HrtA和HrtA的直接宿主衍生的相互作用 Sp50(目标2)。最后，我将使用宿主直接遗传扰动筛选来描述宿主-病原体合成的遗传 与缺乏HrtA或Sp50的帕氏杆菌菌株的相互作用(目标3)。通过这项工作，我不仅将延伸我们的 了解SFG立克次体的发病机制，但也将展示一种合成遗传学的潜力 功能未知的病原体基因的研究和注释方法。这些研究的结果可能 还为治疗方法的发展提供信息，如针对SFG立克次体物种的疫苗。 这个项目将在麻省理工学院进行，麻省理工学院的培训环境非常出色，高度协作。 申请人(Brandon Sit博士)可使用本项目所需的所有设施和设备。培训 该项目附带的计划包括由Rebecca Lamason博士联合指导的Sit博士(主要 赞助商)和保罗·布莱尼(共同赞助商)，旨在定位薛博士过渡到独立的 在这项工作结束时的调查员位置。

PROJECT SUMMARY/ABSTRACT Intracellular bacterial pathogens manipulate host cells through a vast array of mechanisms. Studying these interactions has propelled our understanding of therapeutic development against these pathogens and host cell biology. However, many intracellular pathogens cannot be easily studied due to their obligate nature and resistance to genetic manipulation. These include the spotted fever group (SFG) Rickettsia, which cause a range of potentially severe arthropod-borne human illnesses, including Rocky Mountain spotted fever. The development of new random mutagenesis systems for SFG Rickettsia has propelled studies of these microbes and hinted at the remarkable diversity of unprecedented pathogen innovations in this genus. Recently, our lab performed a small-scale transposon mutagenesis screen in the model rickettsial species R. parkeri to identify attenuated mutants. This screen led to the isolation of >100 R. parkeri mutants with infection defects, with only a few containing insertions in genes previously linked to R. parkeri virulence. The remaining strains represent a valuable tool for probing and understanding R. parkeri and intracellular pathogen biology. Over 15% of the genes hit in this screen are unannotated. Two of these unannotated genes, hrtA and sp50, encode R. parkeri proteins that are predicted to be surface-exposed or secreted and have putative structural features suggestive of direct binding to host proteins. I hypothesize that HrtA and Sp50 are novel R. parkeri secreted or surface-exposed effectors that can hijack specific host functions to promote infection. In this proposal, I will first demonstrate the spatiotemporal niches of both HrtA and Sp50 (Aim 1) to establish how they phenotypically contribute to R. parkeri infection. Then, I will use affinity purification approaches to identify direct host-derived interactors of HrtA and Sp50 (Aim 2). Finally, I will use host-direct genetic perturbation screens to profile host-pathogen synthetic genetic interactions with R. parkeri strains lacking HrtA or Sp50 (Aim 3). Through this work, I will not only extend our understanding of SFG Rickettsia pathogenesis, but will also demonstrate the potential of a synthetic genetic approach for investigating and annotating pathogen genes of unknown function. Results from these studies may also inform development of therapeutics such as vaccines against SFG Rickettsia species. The training environment at MIT, where this project will be carried out, is outstanding and highly collaborative. All facilities and equipment required for this project are available to the applicant (Dr. Brandon Sit). The training plan accompanying this project involves the joint mentorship of Dr. Sit by Drs. Rebecca Lamason (primary sponsor) and Paul Blainey (co-sponsor), and is designed to position Dr. Sit for a transition to an independent investigator position at the end of this work.