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

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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.