Investigating genes of unknown function required for Rickettsia parkeri infection

项目概要/摘要 细胞内细菌病原体通过大量机制操纵宿主细胞。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，许多细胞内病原体由于其专性性质而不容易研究， 对基因操纵的抵抗这些包括斑点热群（SFG）立克次体， 潜在的严重节肢动物传播的人类疾病，包括落基山斑疹热。的 新的SFG立克次体随机诱变系统的开发推动了对这些微生物的研究 并暗示了该属中前所未有的病原体创新的显着多样性。最近，我们的实验室 进行了一个小规模的转座子诱变筛选的模式立克次体物种R。parkeri识别 减毒突变体该筛选导致>100 R的分离。具有感染缺陷的parkeri突变体， 一些含有先前与R.帕克氏菌毒力其余菌株代表 是探索和理解R. Parkeri和细胞内病原体生物学。超过15%的基因 该屏幕中命中是未注释的。这些未注释的基因中的两个，hrtA和sp 50，编码R。帕克氏蛋白 被预测为表面暴露或分泌的，并具有暗示直接 与宿主蛋白质结合。我推测HrtA和Sp 50是新的R. Parkeri分泌或表面暴露 可以劫持特定宿主功能以促进感染的效应器。在本建议中，我将首先演示 HrtA和Sp 50的时空小生境（目的1），以确定它们如何表型地有助于R。帕克里 感染然后，我将使用亲和纯化方法来鉴定HrtA的直接宿主衍生的相互作用物， Sp 50（目标2）。最后，我将使用宿主直接遗传干扰筛选来分析宿主-病原体合成遗传干扰。 与R的相互作用缺乏HrtA或Sp 50的parkeri菌株（Aim 3）。通过这项工作，我不仅将扩大我们的 了解SFG立克次体的发病机制，但也将证明合成遗传的潜力， 一种研究和注释功能未知的病原体基因的方法。这些研究的结果可能 还为治疗剂如针对SFG立克次体物种的疫苗的开发提供了信息。 该项目将在麻省理工学院开展，该学院的培训环境非常出色，而且具有高度的协作性。 申请人（布兰登薛博士）可使用本项目所需的所有设施和设备。培训 伴随这个项目的一个计划包括由Rebecca Lamason博士（小学）联合指导Sit博士 发起人）和Paul Blainey（共同发起人），旨在定位薛博士过渡到独立 调查员在这项工作的最后。

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.