Diarrheal pathogens and the evolution of intestinal signaling

美国每年有1.79亿人患有感染性腹泻，自1980年以来死亡人数增加了四倍。在美国感染性腹泻的一个主要原因是产肠毒素E.大肠杆菌，其产生热稳定肠毒素（ST）以过度激活宿主鸟苷酸环化酶C（GC-C），鸟苷酸环化酶C是肠道中液体平衡的主要调节剂。ST在宿主中引起腹泻，通过促进传播到新宿主而使细菌受益。该团队的成员先前将ST-GC-C界面确定为宿主和病原体之间持续的进化冲突，其中GC-C在灵长类动物，特别是蝙蝠中进行正向选择。后一分类群还呈现GC-C的宿主配体尿鸟苷素的补偿性进化，维持其激活GCC的能力 尽管GC-C响应细菌进化压力而进化。在这项提案中，这种意想不到的补偿性演变的关键生理途径将被利用，以确定潜在的抑制剂的ST作为一种治疗策略，在人类感染性腹泻。 中心假设是尿鸟苷素和ST的快速进化导致GC-C的结合和活化的功能差异，并且可以发现竞争性抑制人类致病性肠毒素引起腹泻的高亲和力-低活性肽，并在未来利用其治疗感染性腹泻。目的（1）研究尿鸟苷素和肠毒素变异对GC-C结合和活化的影响。将进行GC-C配体的进化分析以鉴定正选择下的位点，并将测试配体的结合和活化以鉴定高亲和力和低活性候选物。目的（2）将鉴定在大肠杆菌感染的细胞和类器官模型中调节GC-C活化的肽。将在竞争测定中测试高亲和力-低活性肽，以确定是否可以阻断肠毒素引起腹泻，作为治疗感染性腹泻的直接方法。这些结果将为未来肽的设计提供信息 感染性腹泻的治疗方法，通过使用靶向治疗而不是主要治疗症状，将大大改善临床护理。该项目将提供进化分析，生化测定和类器官模型方面的优秀多学科培训，并辅以传染病的临床指导和阴影。培训计划的制定是为了完成进行独立，合作研究的中心目标;提高沟通和赠款写作技能;学习如何有效地指导和教导学员， 环境;并磨练临床技能，为返回医学院做准备。这项培训是理想的未来的医生，科学家与改善病人护理的基础研究和儿科传染病的临床治疗的交叉点的目标。

179 million people in the US suffer from infectious diarrhea every year, and deaths have increased four times since 1980. A major cause of infectious diarrhea in the US is enterotoxigenic E. coli, which produces heatstable enterotoxins (ST) to overactivate host guanylyl-cyclase C (GC-C), a major regulator of fluid balance in the gut. STs cause diarrhea in the host, benefiting the bacteria by facilitating spread to new hosts. Members of this team previously identified the ST-GC-C interface as an ongoing evolutionary conflict between hosts and pathogens, where GC-C was undergoing positive selection in primates and especially bats. The latter taxa also present compensatory evolution in the host ligand for GC-C, uroguanylin, maintaining its ability to activate GCC despite the evolution of GC-C in response to the bacterial evolutionary pressure. In this proposal, this unexpected compensatory evolution in a critical physiologic pathway will be leveraged to identify potential inhibitors of STs as a treatment strategy for infectious diarrhea in humans. The central hypothesis is that rapid evolution of uroguanylin and ST resulted in functional differences in binding and activation of GC-C and high affinity-low activity peptides that will competitively inhibit human-pathogenic enterotoxins from causing diarrhea can be found and leveraged to treat infectious diarrhea in the future. Aim (1) will characterize the effect of uroguanylin and enterotoxin variation on binding and activation of GC-C. Evolutionary analyses of GC-C ligands will be conducted to identify sites under positive selection, and ligands will be tested for binding and activation to identify high affinity and low activity candidates. Aim (2) will identify peptides that modulate GC-C activation in cellular and organoid models of diarrheal infection. High affinity-low activity peptides will be tested in competition assays to determine if enterotoxins can be blocked from causing diarrhea as a direct method of treating infectious diarrhea. The results will inform the design of future peptide treatments for infectious diarrhea that will greatly improve clinical care by using targeted therapy instead of mainly treating symptoms. This project will provide excellent multidisciplinary training in evolutionary analysis, biochemical assays, and organoid models, complemented by clinical mentorship and shadowing in infectious disease. The training plan was developed to complete the central goals of conducting independent, collaborative research; advancing communication and grant-writing skills; learning how to effectively mentor and teach trainees in a supportive environment; and honing clinical skills in preparation for the return to medical school. This training is ideal for a future physician-scientist with the goal of improving patient care at the intersection of basic research and clinical treatment of pediatric infectious diseases.