Identifying Factors That Control Germinant Sensitivity During Clostridium Difficile Spore Germination

孢子萌发对于主要医院病原体艰难梭菌启动和传播感染至关重要，但临床分离株对胆汁盐萌发体的反应差异很大。这种变异的临床意义目前尚不清楚，但最近的报告表明，对胆盐萌芽牛磺胆酸盐敏感性降低的菌株可能更有可能引起严重的疾病。确定发芽敏感性变化是否影响疾病严重程度的一个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差异使识别这些因素变得复杂，因此我们将使用靶向诱变和遗传筛选来识别增加或减少艰难梭菌萌芽敏感性的关键调节因子。这些调节剂对萌发敏感性、萌发动力学和萌发异质性的影响将使用创新的拉曼光谱和微分干涉显微镜（DIC）方法来确定，该方法可以在单细胞水平上同时测量孢子萌发过程中的关键事件，与现有方法不同。通过确定调节发芽敏感性的因素，拟议的研究将为艰难梭菌如何感知肠道环境以引发感染提供关键见解，指导开发改变特定胆汁盐水平的治疗策略，并可能揭示新的治疗靶点。此外，在拟议的工作中开发的同基因菌株将允许在未来的研究中在感染动物模型中严格测试萌发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.