Investigating genes of unknown function required for Rickettsia parkeri infection

研究帕氏立克次体感染所需的未知功能基因

基本信息

批准号：
10724245
负责人：
Brandon Yiu Chung Sit
金额：
$ 6.95万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2025-08-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10724245
关键词：
Adhesions Affinity Chromatography Antigens Attenuated Autophagocytosis Binding Bioinformatics Biology CRISPR/Cas technology Cells Cellular biology Collagen Complex Cytoskeleton Cytosol Defect Development Diagnostic Dissection Engineering Environment Equipment Fimbriae Proteins Genes Genetic Genetic Screening Genome Genomics Goals Human Imaging Techniques Infection Integration Host Factors Invaded Investigation Joints Knock-out Knowledge Libraries Life Cycle Stages Life Style Link Lipoproteins Mentorship Methods Microbe Modeling Mutagenesis Nature Pathogenesis Pathogenicity Pathway interactions Phenotype Positioning Attribute Probability Protein Secretion Proteins Public Health Reporting Research Personnel Resistance Rickettsia Rickettsia Infections Rickettsia parkeri Rocky Mountain Spotted Fever Role Surface System Techniques Therapeutic Time Training Vaccines Vascular Diseases Virulence Work arthropod-borne design gene product genetic approach genetic manipulation improved innovation mutant novel novel therapeutics pathogen pathogenic bacteria receptor binding screening spatiotemporal spotted fever therapeutic development tool

项目摘要

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.

项目概要/摘要细胞内细菌病原体通过多种机制操纵宿主细胞。研究这些相互作用促进了我们对针对这些病原体和宿主细胞的治疗开发的理解生物学。然而，许多细胞内病原体由于其专性性质和对基因操纵的抵抗力。其中包括斑点热病群 (SFG) 立克次体，它会引起一系列潜在的严重节肢动物传播的人类疾病，包括落基山斑疹热。这针对 SFG 立克次体的新型随机诱变系统的开发推动了对这些微生物的研究并暗示该属前所未有的病原体创新具有显着的多样性。最近，我们实验室在模型立克次体物种 R. parkeri 中进行了小规模转座子诱变筛选，以鉴定减毒突变体。该筛选分离出超过 100 个具有感染缺陷的 R. parkeri 突变体，仅一些包含先前与帕克里氏菌毒力相关的基因的插入。其余菌株代表探索和了解帕克里氏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注释。其中两个未注释的基因，hrtA 和 sp50，编码 R. parkeri 蛋白预计是表面暴露或分泌的，并且具有暗示直接的推定结构特征与宿主蛋白结合。我假设 HrtA 和 Sp50 是 R. parkeri 分泌的或表面暴露的新型物质可以劫持特定宿主功能以促进感染的效应器。在这个提案中，我将首先展示 HrtA 和 Sp50 的时空生态位（目标 1）以确定它们如何对 R. parkeri 做出表型贡献感染。然后，我将使用亲和纯化方法来识别 HrtA 和 HrtA 的直接宿主衍生相互作用因子 Sp50（目标 2）。最后，我将使用宿主直接遗传扰动筛选来分析宿主-病原体合成遗传与缺乏 HrtA 或 Sp50 的 R. parkeri 菌株相互作用（目标 3）。通过这项工作，我不仅会扩展我们的了解 SFG 立克次体发病机制，但也将展示合成遗传的潜力研究和注释未知功能的病原体基因的方法。这些研究的结果可能还为治疗方法的开发提供信息，例如针对 SFG 立克次体物种的疫苗。该项目将在麻省理工学院进行，其培训环境非常出色且高度协作。本项目所需的所有设施和设备均可供申请人（薛布兰登博士）使用。培训内容该项目的配套计划涉及薛博士和 Drs. 的联合指导。丽贝卡·拉马森（小学发起人）和保罗·布莱尼（共同发起人），旨在使薛博士过渡到独立这项工作结束时的调查员职位。