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

• 批准号: 10535336
• 负责人: Brandon Yiu Chung Sit
• 金额: $ 6.72万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 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/10535336
• 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; Investigation; Joints; Knock-out; Knowledge; Libraries; Life Style; Link; Lipoproteins; Mentorship; Methods; Microbe; Modeling; Mutagenesis; Nature; Pathogenesis; Pathogenicity; Pathway interactions; Phenotype; Positioning Attribute; Proteins; Public Health; Reporting; Research Personnel; Resistance; Rickettsia; Rickettsia Infections; Rickettsia parkeri; Rocky Mountain Spotted Fever; Role; Suggestion; Surface; System; Techniques; Therapeutic; Time; Training; Vaccines; Vascular Diseases; Virulence; Work; arthropod-borne; base; 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

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.

项目摘要/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这些病原体和宿主细胞的治疗发展的理解 生物学。但是，许多细胞内病原体由于其专有性质而无法轻松研究 对遗传操作的抵抗力。其中包括斑点发烧组（SFG）立克，这会导致一个范围 潜在的严重节肢动物传播的人类疾病，包括落基山斑点发烧。这 开发用于SFG人力素的新的随机诱变系统已推动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 在型号里克西亚物种R. parkeri中进行了小规模的转座子诱变屏幕以识别 衰减突变体。此屏幕导致> 100 R. parkeri突变体的隔离，只有感染缺陷，只有 一些包含与R. parkeri毒力相关的基因中的插入。其余应变代表 探测和了解R. parkeri和细胞内病原体生物学的有价值工具。超过15％的基因 在此屏幕中击中未被注释。这些未注释的基因中有两个HRTA和SP50编码R. Parkeri蛋白 预计将表面暴露或分泌，并具有推定的结构特征 与宿主蛋白结合。我假设HRTA和SP50是新颖的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。在此建议中，我将首先证明 HRTA和SP50的时空壁ni（AIM 1），以确定它们表型对R. parkeri的贡献 感染。然后，我将使用亲和力纯化方法来识别HRTA和 SP50（AIM 2）。最后，我将使用宿主直接遗传扰动筛选来介绍宿主 - 道原成遗传遗传 与缺乏HRTA或SP50的R. parkeri菌株的相互作用（AIM 3）。通过这项工作，我不仅会扩展我们的 了解SFG人力素的发病机理，但也将证明合成遗传的潜力 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 还可以告知针对SFG人力素物种的疫苗等治疗剂的开发。 该项目将在该项目进行的培训环境非常出色且高度协作。 该项目所需的所有设施和设备均可申请人（Brandon Sit博士）。培训 该项目伴随的计划涉及Sit博士的联合指导。丽贝卡·拉马森（Rebecca Lamason）（小学 赞助商）和保罗·布莱尼（Paul Blainey）（共同发起人），旨在定位Sit博士，以过渡到独立 研究人员在这项工作结束时的职位。