The macrophage Repairome

巨噬细胞修复组

基本信息

批准号：
10448493
负责人：
Stephanie M M Seveau
金额：
$ 7.88万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-12 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10448493
关键词：
Bacterial Toxins Binding CRISPR library CRISPR screen CRISPR/Cas technology Candidate Disease Gene Cell Line Cell Maintenance Cell Separation Cell membrane Cells Chimeric Proteins Cholesterol Communicable Diseases Complex Cytosol Defense Mechanisms Development Exposure to Family Fluorescence Fluorescent Dyes Funding Future Generations Genes Genomic DNA Genomic Library Goals Gram-Positive Bacteria Grant Guide RNA Homeostasis Hospitalization Human Immune Immune response Immunity Infection Knock-out Libraries Listeria monocytogenes Listeria monocytogenes hlyA protein Listeriosis Literature Membrane Methods Microbe Ontology Outcome Pathogenesis Pathway Analysis Pathway interactions Perforation Phagosomes Pharmacology Phenotype Plasma Cells Play Population Proteins Research Role Small Interfering RNA Targeted Toxins Therapeutic Intervention Toxin Validation Virulence Factors Work antimicrobial base cell injury cost deep sequencing experience foodborne pathogen genome editing genome-wide inhibitor live cell imaging macrophage monocyte new therapeutic target next generation sequencing novel pathogen pathogenic bacteria prevent repaired screening spatiotemporal therapeutic development tool whole genome

项目摘要

Summary Plasma membrane disruption by pore-forming toxins (PFTs) is a most common and ancient strategy used by bacterial pathogens to infect their host and evade the host’s immune responses. Listeriolysin O (LLO) is a PFT produced by the foodborne pathogen Listeria monocytogenes (Lm). Lm is a Gram-positive bacterium responsible for listeriosis, a severe illness leading to 99% hospitalization and up to 30% fatality despite treatment. Lm is a facultative intracellular pathogen that infects a large array of cells including macrophages. Although Lm produces numerous virulence factors, LLO is uniquely known to be indispensable for pathogenesis. Therefore, LLO and the host pathways targeted by this toxin are promising targets for the development of therapeutic interventions. LLO is secreted at all stages of the Lm intracellular lifecycle and binds cholesterol to assemble a large transmembrane pore complex. This virulence factor has long been known to perforate the membrane of the Lm- containing phagosome to release Lm into its replicative niche, the cytosol. It was recently established that LLO also perforates the host cell plasma membrane, which facilitates cell invasion and phagosomal escape. Monocyte/Macrophages, which specialize in the capture and destruction of microbes, evolved cytoprotective mechanisms (referred to as the macrophage repairome) to maintain cell homeostasis and survival despite LLO attack. How macrophages repair their plasma membrane and prevent toxin attack is not fully understood. The goal of this R03 proposal is to develop tools to discover the “macrophage repairome” using unbiased whole- genome screening. Our lab successfully developed fluorescence-based screening methods to analyze the repair machineries of cells exposed to LLO. To establish the macrophage repairome in an unbiased fashion, we will perform a whole-genome screen using CRISPR/Cas9 genome editing. We will generate a CRISPR/Cas9 library in THP-1 cells (human monocyte-like cell line) and screen the library for cells unable to maintain their integrity upon LLO exposure using florescence-activated cell sorting (FACS)-selectable phenotype (positive screen). Indeed, damaged cells with deficient cell repair will be fluorescent, whereas intact cells will exclude the fluorescent dye. Deep sequencing will generate a list of candidate genes required for maintaining macrophage integrity. We will perform pathway analysis, select the most novel and promising pathways, and validate the selected pathways in THP-1 cells. These pathways will be studied in detail in the context of L. monocytogenes infection via future R01 funding. Understanding the mechanisms used by macrophages to counteract bacterial pore-forming toxins is expected to facilitate the development of novel antimicrobial treatments to alleviate the burden of infectious diseases.

概括通过成孔毒素（PFT）破坏质膜是一种最常见和最古老的策略细菌病原体感染宿主并逃避宿主的免疫反应。由食源性病原体单核细胞增生李斯特氏菌 (Lm) 产生，是一种革兰氏阳性细菌。对于李斯特菌病来说，尽管接受治疗，仍会导致 99% 的人住院治疗，死亡率高达 30%。兼性细胞内病原体，可感染大量细胞，包括巨噬细胞，但会产生 Lm。 LLO 与众多毒力因子一起被认为是发病机制中不可缺少的。这种毒素针对的宿主途径是开发治疗干预措施的有希望的目标。 LLO 在 Lm 细胞内生命周期的所有阶段都会分泌，并与胆固醇结合以组装一个大的人们早就知道这种毒力因子可以穿透 Lm- 的膜。最近确定 LLO 含有吞噬体，可将 Lm 释放到其复制生态位（胞质溶胶）中。它还会穿透宿主细胞质膜，从而促进细胞侵袭和吞噬体逃逸。单核细胞/巨噬细胞专门捕获和破坏微生物，进化出了细胞保护能力尽管 LLO 仍维持细胞稳态和存活的机制（称为巨噬细胞修复组）巨噬细胞如何修复质膜并防止毒素攻击尚不完全清楚。该 R03 提案的目标是开发工具，使用无偏整体发现“巨噬细胞修复组” 我们的实验室成功开发了基于荧光的筛选方法来分析修复。为了以公正的方式建立巨噬细胞修复组，我们将使用暴露于 LLO 的细胞机器。使用 CRISPR/Cas9 基因组编辑进行全基因组筛选我们将生成 CRISPR/Cas9 文库。在 THP-1 细胞（人类单核细胞样细胞系）中筛选无法维持完整性的细胞在 LLO 暴露后，使用荧光激活细胞分选 (FACS) 可选择表型（阳性筛选）。事实上，细胞修复缺陷的受损细胞会发出荧光，而完整的细胞将排除荧光染料深度测序将生成维持巨噬细胞所需的候选基因列表。我们将进行路径分析，选择最新颖和最有前途的路径，并验证该路径。 THP-1 细胞中的选定途径将在单核细胞增生李斯特菌的背景下进行详细研究。通过未来的 R01 资助了解巨噬细胞抵抗细菌的机制。成孔毒素有望促进新型抗菌疗法的开发，以缓解传染病的负担。