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The macrophage Repairome

巨噬细胞修复组

基本信息

批准号：
10294333
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 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10294333
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/antagonist 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.

总结