Project 3 - Human colonoids as a model for the pathobiology of EHEC

摘要 拟议的研究将促进对由食源性志贺毒素引起的肠道疾病的了解。 产生肠出血性大肠杆菌(EHEC)，这是威胁生命的出血性结肠炎的主要原因 以及美国的溶血性尿毒症综合征。而志贺毒素2a是肠道和肠病毒的主要毒力因子。 肠外疾病的表现，临床资料强烈提示STX-中毒血症发生在疾病早期。 而且是短暂的。因此，我们的研究将集中在EHEC-宿主的早期相互作用，特别是复合体 肠出血性大肠杆菌与人结肠细胞、杯状细胞和包括巨噬细胞在内的免疫细胞的相互作用 还有中性粒细胞。我们令人兴奋的初步数据是使用一种新的EHEC感染模型获得的，称为 人结肠类单层(HCM)，即来源于成人干细胞的原代结肠上皮细胞培养物 从健康捐赠者的结肠活检中分离出来。在分化时，HCM由所有主要类型的 上皮细胞，包括结肠细胞、杯状细胞和肠内分泌细胞。它们发育成熟的微绒毛和 产生一层厚厚的粘液，类似于通常存在于人类结肠中的粘液。使用这些HCM 我们在这里证明，EHEC感染会导致粘液层的破坏，从而使EHEC 可以接触到结肠细胞表面。丝氨酸蛋白酶ESPP在EHEC的定植中起作用，而且 诱导离子分泌，提示其在EHEC所致水样腹泻中的作用。这些数据表明，人类 结肠腺样体概括了EHEC感染，并提供了不同于其他致病机制的独特见解 在人类结肠癌细胞系中进行的研究，使人们能够更好地认识毒力的作用 新的治疗靶点的影响因素和评价。与这种病原体有关，我们将检验假设 唯一代表体外人类结肠上皮的HCM，概括了人类EHEC感染和 在发病机制方面提供独特的见解。检验这一假说，增进我们对EHEC的了解 针对肠道发病机制，我们提出了以下研究目标：1.确定丝氨酸蛋白酶ESPP在肠道发病机制中的作用 肠出血性大肠杆菌在人结肠上皮细胞的定植和毒力发展。2.确定分子 肠出血性大肠杆菌引起水样腹泻的机制。3.确定巨噬细胞和中性粒细胞在 EHEC诱导的免疫反应。建议中的实验结果将进一步阐明 EHEC与人结肠上皮相互作用的分子机制，测试ESPP丝氨酸的作用 蛋白水解酶在肠出血性肠杆菌定植和水样腹泻发展中的作用以及中性粒细胞的建立 和巨噬细胞清除EHEC。

Abstract The proposed studies will advance the understanding of enteric disease caused by food-borne, Shiga-toxin- producing enterohemorrhagic E. coli (EHEC), whichis the major cause of life-threatening hemorrhagic colitis and hemolytic uremic syndrome in the US. While Shiga toxin 2a is the main virulence factor for intestinal and extra-intestinal disease manifestations, clinical data strongly suggest that Stx-toxemia occurs early in illness and is short-lived. Thus, our studies will focus on early EHEC-host interactions, particularly the complex interaction between EHEC and the human colonocyte, goblet cells and immune cells, including macrophages and neutrophils. Our exciting preliminary data are gained using a novel model of EHEC infection, termed human colonoid monolayers (HCM), which are primary colonic epithelial cultures derived from adult stem cells isolated from colonic biopsies of healthy donors. Upon differentiation, HCM are composed of all major types of epithelial cells, including colonocyte, goblet and enteroendocrine cells. They develop mature microvilli and produce a thick layer of mucus similar to that which is normally present in the human colon. Using these HCM we are demonstrating here that EHEC infection results in the destruction of the mucus layer allowing EHEC to gain access to the colonocyte surface. The serine protease EspP plays a role in EHEC colonization and also induces the ion secretion, indicating its role in EHEC-induced watery diarrhea. These data suggest that human colonoids recapitulate EHEC infection and provide unique insights into pathogenesis that differ from other studies performed in human colon cancer cell lines, allowing improved appreciation of the roles of virulence factors and assessment of novel therapeutic targets. Relevant to this pathogen, we will test the hypothesis that HCM, which uniquely represent ex vivo human colonic epithelium, recapitulate human EHEC infection and provide unique insights in pathogenesis. To test this hypothesis and enhance our understanding of EHEC intestinal pathogenesis, we propose the following Aims: 1. Determine the role of serine protease EspP in EHEC colonization of human colonic epithelium and virulence development. 2. Determine the molecular mechanisms of EHEC induced watery diarrhea. 3. Determine the role of macrophages and neutrophils in EHEC-induced immune response. The results gained from the proposed experiments will further elucidate the molecular mechanisms for EHEC interactions with human colonic epithelium, test the role of EspP serine protease in EHEC colonization and development of watery diarrhea as well as establish the role of neutrophil and macrophages in EHEC clearance.