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

艰难梭菌是一种形成孢子的细菌病原体，是医疗保健相关疾病的主要原因。 在美国感染。而C.人们早就知道艰难梭菌产生强效细胞毒素的能力， 使其引起炎性结肠炎，但对C.使其能够 在竞争激烈的肠道环境中成长和生存。像许多肠道细菌一样，C。艰难梭菌产生表型 在一个看似克隆的种群中有不同的亚种群。这一观察结果导致了一个假设，即C。 艰难梭菌利用表型异质性来促进其在动态肠道环境中的存活。不幸的是， 由于缺乏跟踪特定亚细胞命运的方法， 人口随着时间的推移。延时显微镜传统上被用来解决这个问题，但现有的 方法不能用于研究C.因为它不能在大气中生长， 氧气我们克服了这一技术挑战，开发了一个简单的厌氧时间推移， 显微镜下观察C.单细胞水平的艰难生长。通过将此方法与 自动谱系跟踪管道，我们已经测量了单个C的生长特性。艰难细胞 第一次这些分析表明，C.艰难梭细胞的大小和延伸率在生长过程中受到严格控制， 在丰富的培养基中生长，但它们在生理应激的存在下变得明显异质。到 了解C。difficile在单细胞水平上适应不同的生理应激源，该提议将 使用新的厌氧成像报告链接基因表达谱的个别细胞，他们的细胞命运。 我们也将使用这个系统来研究C。艰难孢子长入营养细胞，并测试是否长出 细胞比营养细胞更容易受到生理压力的影响，这一问题尚未得到研究。 在任何一个系统中都是如此。在C语言中确定这些问题的答案。difficile将告知策略 抑制C.艰难的感染，而本提案中建立的方法将具有广泛的实用性， 研究其他厌氧菌的生理学

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.