Mechanisms underlying diarrhea and gut inflammation mediated by Enterotoxigenic and Enteropathogenic E. coli

项目总结 产热耐药传染性肠毒素大肠埃希菌(ETEC)介导的腹泻病 肠毒素(ST)是发展中国家发病率的重要原因之一，5岁以下儿童 是受影响最严重的。感染这些毒株的儿童表现出中到重度的腹泻，但面临 最大的长期发病率和增加的死亡风险。目前还没有直接的疗法来治疗 稳定的毒素介导的腹泻，但口服锌盐已被证明是有益的。 这一提议的长期目标是阐明慢性cGMP驱动的机制 增加对肠道细菌病原体的敏感性。ST与细胞表面受体鸟苷酸环化酶C结合 (GC-C，由GUCY2C编码)，在肠道的所有细胞类型中表达，但不在免疫细胞中表达。我们 先前描述和表征了GUCY2C和产生的敲入小鼠的过度激活突变 表现为肠道cGMP升高和腹泻。我们的研究表明，这些小鼠表现出类似MSD的症状 表型。因此，我们假设这些小鼠可以作为了解肠道的临床前模型。 在ST介导的腹泻持续发作的儿童中可见的环境。这些小鼠显示出一种干扰素- 驱动肠道基因特征和潘氏细胞缺陷。我们建议实现以下目标和 本提案中的问题：(1)阐明转基因中潘氏细胞功能障碍的机制 利用基因工程细胞系和有机培养在分子水平上发生小鼠；(2)研究机制 转基因小鼠的STAT1诱导和炎症方面；(3)监测STAT1的招募和成熟 肠相关淋巴组织(GALT)在cGMP介导的腹泻中的特征和(4)询问是否 腹泻的流行状况会导致对肠源性E. Coli(EPEC)。众所周知，口服硫酸锌可以缩短受影响儿童的腹泻发作时间。 因此，我们将在动物体内加入锌的管理，试图逆转炎症和其他 在转基因小鼠中看到的有害表型。 总的来说，使用这些转基因小鼠来了解传染性分泌性腹泻不仅会 阐明肠道在长期分泌性腹泻期间发生的潜在变化，但也揭示了靶点 用于缓解症状的治疗(例如，锌)。此外，我们的研究将确定可能 要有针对性，以确保与另一种细菌病原体混合感染的影响最小。

PROJECT SUMMARY Diarrheal diseases mediated by infectious enterotoxigenic Escherichia coli (ETEC) that produce heat-stable enterotoxins (ST) are one of the significant causes of morbidity in developing countries, with children below 5 being the most severely affected. Children infected with these strains show moderate-to-severe diarrhea but face the greatest long-term morbidity and increased risk of death. There are currently no directed therapies against stable toxin-mediated diarrhea, but oral administration of zinc salts has proven to be beneficial. The long-term goal of this proposal is to elucidate the mechanisms of how chronic cGMP-driven increases susceptibility to enteric bacterial pathogens. ST binds to the cell surface receptor guanylyl cyclase C (GC-C, encoded by GUCY2C), expressed in all cell types in the gut but not immune cells. We previously described and characterized hyperactivating mutations in the GUCY2C and generated knock-in mice that display elevated intestinal cGMP and diarrhea. Our studies show that these mice display MSD-like phenotypes. Therefore, we hypothesize that these mice could serve as a pre-clinical model to understand the gut environment seen in children with prolonged episodes of ST-mediated diarrhea. These mice show an interferon- driven intestinal gene signature and Paneth cell defects. We propose to pursue the following objectives and questions in this proposal: (1) Elucidate mechanisms by which the Paneth cell dysfunction seen in transgenic mice occurs at the molecular level using engineered cell lines and organoid cultures; (2) study mechanistic aspects of Stat1 induction, and inflammation in transgenic mice; (3) Monitor the recruitment and maturation of the gut-associated lymphoid tissue (GALT) during cGMP-mediated diarrhea characterize and (4) ask whether a prevailing condition of diarrhea induces a more severe response to further infection by enteropathogenic E. coli (EPEC). Oral administration of zinc sulfate is known to shorten diarrheal episodes in affected children. Therefore, we will incorporate zinc administration in animals to attempt to reverse inflammation and other detrimental phenotypes seen in transgenic mice. Collectively, using these transgenic mice to understand infectious secretory diarrhea will not only elucidate the underlying changes that occur in the gut during prolonged secretory diarrhea but also reveal targets for therapy (e.g., zinc) in alleviating symptoms. Furthermore, our studies will identify vulnerabilities that could be targeted to ensure that co-infection with another bacterial pathogen has minimal impact.