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Linking Gene Expression Profiles to Cell Fate in Clostridioides difficile Using Time-Lapse Microscopy

使用延时显微镜将基因表达谱与艰难梭菌细胞命运联系起来

基本信息

批准号：
10330034
负责人：
Aimee Shen
金额：
$ 24.36万
依托单位：
TUFTS UNIVERSITY BOSTON
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：
2021-01-15 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10330034
关键词：
Address Anaerobic Bacteria Animal Model Antibiotics Bacteria Bacterial Physiology Cell Size Cell Survival Cells Clinical Clostridium difficile Coupling Cytotoxin Data Drug Metabolic Detoxication Environment Gastroenteritis Gene Expression Gene Expression Profile Genes Genetic Transcription Growth Healthcare Systems Heme Heterogeneity Image Immune Individual Infection Inflammatory Kinetics Lead Length Link Measures Methodology Methods Microscopy Monitor Muramidase Nosocomial Infections Oxygen Pharmacology Phase Phenotype Physiological Physiology Population Predisposition Production Property Reagent Reporter Reproduction spores Resistance Ribotypes Stress System Testing Time Toxin United States Variant Virulence Work acute infection analysis pipeline antimicrobial peptide base biological adaptation to stress cell behavior cost diarrheal disease gut bacteria gut microbiota healthcare-associated infections imaging modality improved innovation insight microbiome components novel pathogen pathogenic bacteria physiologic stressor response single cell analysis stressor time use

项目摘要

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of healthcare-associated infections in the United States. While C. difficile’s ability to produce potent cytotoxins has long been known to allow it to cause inflammatory diarrheal disease, little is known about the properties of C. difficile that allow it to grow and survive in the competitive gut environment. Like many gut bacteria, C. difficile generates phenotypically distinct sub-populations within a seemingly clonal population. This observation has led to the hypothesis that C. difficile uses phenotypic heterogeneity to promote its survival in the dynamic gut environment. Unfortunately, testing this hypothesis has been complicated by the absence of methods for following the fate of specific sub- populations over time. Time-lapse microscopy has traditionally been used to address this question, but existing methods cannot be used to study the growth of C. difficile because it cannot grow in the presence of atmospheric oxygen. We have overcome this technological challenge by developing a simple anaerobic time-lapse microscopy method for visualizing C. difficile growth at the single-cell level. By combining this method with an automated lineage tracking pipeline, we have measured the growth properties of individual C. difficile cells for the first time. These analyses indicate that C. difficile cell size and elongation rates are tightly controlled during growth in rich media, but they become markedly heterogeneous in the presence of physiological stress. To understand how C. difficile adapts to different physiological stressors at the single-cell level, this proposal will use novel anaerobic imaging reporters to link the gene expression profile of individual cells to their cellular fate. We will also use this system to study C. difficile spore outgrowth into vegetative cells and test whether outgrowing cells are more vulnerable to physiological stressors than vegetative cells, a question that has not yet been studied in any system to our knowledge. Determining the answers to these questions in C. difficile will inform strategies for inhibiting C. difficile infections, while the methods established in this proposal will have broad utility for studying the physiology of other anaerobes.

艰难梭菌是一种芽孢形成的细菌病原体，是医疗保健相关的主要原因