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

艰难梭状芽胞杆菌是一种形成孢子的细菌病原体，是与医疗保健相关的 在美国的感染。虽然艰难梭菌产生强效细胞毒素的能力早已为人所知 允许它引起炎症性腹泻，但人们对艰难梭菌的特性知之甚少。 在充满竞争的胆识环境中成长和生存。像许多肠道细菌一样，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C。 艰难梭菌利用表型异质性促进其在动态肠道环境中的生存。不幸的是， 由于缺乏追踪特定亚类命运的方法，检验这一假说变得复杂起来。 随着时间的推移种群数量。时间推移显微镜传统上被用来解决这个问题，但现有的 方法不能用来研究艰难梭菌的生长，因为它不能在大气中生长。 氧气。我们已经克服了这一技术挑战，开发了一种简单的厌氧延时 在单细胞水平上观察艰难梭菌生长的显微镜方法。通过将此方法与 自动谱系跟踪流水线，我们测量了艰难梭菌单个细胞的生长特性 第一次。这些分析表明艰难梭菌的细胞大小和延长率在 在富介质中生长，但在生理胁迫下变得明显的异质性。至 了解艰难梭菌如何在单细胞水平上适应不同的生理应激源，这项建议将 使用新的厌氧成像报告将单个细胞的基因表达谱与它们的细胞命运联系起来。 我们还将使用该系统来研究艰难梭菌孢子向营养细胞的生长，并测试是否超过生长 细胞比营养细胞更容易受到生理压力的影响，这个问题还没有被研究过。 据我们所知，在任何系统中。在艰难梭菌中确定这些问题的答案将为战略提供参考 用于抑制艰难梭菌感染，而本提案中建立的方法将对 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.