Identifying Factors That Control Germinant Sensitivity During Clostridium Difficile Spore Germination

孢子萌发是主要医院病原体艰难梭菌启动和传播感染所必需的，但临床分离株对胆盐萌发剂的反应性差异很大。这种变异的临床意义目前尚不清楚，尽管最近的报告表明，对胆盐萌发剂牛磺胆酸盐敏感性降低的菌株可能更容易引起严重疾病。确定发芽敏感性的变化是否影响疾病严重程度的关键障碍是缺乏控制发芽敏感性的已知因素。由于临床分离株之间的广泛变异使鉴定这些因子变得复杂，我们将使用靶向诱变和遗传筛选来鉴定增加或减少C.难以萌发的敏感性。这些监管机构的发芽敏感性，发芽动力学和发芽异质性的影响将确定使用创新的拉曼光谱和微分干涉显微镜（DIC）的方法，可以同时测量在单细胞水平孢子萌发过程中的关键事件，不同于现有的方法。通过确定调节萌发敏感性的因素，拟议的研究将提供关键的见解，如何C。艰难梭菌感知肠道环境，以启动感染，指导改变特定胆汁盐水平的治疗策略的发展，并可能揭示新的治疗靶点。此外，在拟议的工作中开发的等基因菌株将允许在未来的研究中在感染的动物模型中严格测试萌发剂敏感性对疾病严重程度的影响。

Spore germination is essential for the major nosocomial pathogen Clostridium difficile to initiate and transmit infection, yet clinical isolates vary widely in their responsiveness to bile salt germinants. The clinical significance of this variation is currently unknown, although recent reports suggest that strains with reduced sensitivity to the bile salt germinant taurocholate may be more likely to cause severe disease. A critical barrier to determining whether variation in germinant sensitivity impacts disease severity is the absence of known factors that control germinant sensitivity. Since identifying such factors is complicated by the extensive variation between clinical isolates, we will use targeted mutagenesis and genetic screens to identify key regulators that either increase or decrease C. difficile germinant sensitivity. The effect of these regulators on the germinant sensitivity, germination kinetics and germination heterogeneity will be determined using an innovative Raman spectroscopy and differential interference microscopy (DIC) method that can simultaneously measure key events during spore germination at the single-cell level, unlike existing methods. By identifying factors that regulate germinant sensitivity, the proposed studies will provide key insights into how C. difficile senses the gut environment in order to initiate infection, guide the development of therapeutic strategies that alter the levels of specific bile salts, and may reveal new therapeutic targets. Furthermore, the isogenic strains developed in the proposed work will allow the impact of germinant sensitivity on disease severity to be rigorously tested in animal models of infection in future studies.