Role of Mucus in EHEC colonization of human colonoids and intestinal disease devleopment

总结 拟议的工作将促进对食源性志贺毒素引起的肠道疾病的了解， 产生肠出血性E.大肠杆菌（EHEC）构成了美国危及生命的出血性大肠杆菌的主要原因。 结肠炎和溶血性尿毒综合征（HUS）。滋贺毒素2a是志贺病毒的主要毒力因子， 肠道和肠外疾病的表现，临床数据强烈表明，Stx毒血症发生 早期患病，寿命短。因此，我们专注于早期EHEC-宿主相互作用，特别是复合物 肠出血性大肠杆菌和肠粘液之间的相互作用。肠粘液代表了抵抗的第一道防线 是肠出血性大肠杆菌与人结肠上皮相互作用的第一个位点。我们 令人兴奋的初步数据表明粘液在人结肠的EHEC定殖中起重要作用。 这些数据是使用一种新的肠出血性大肠杆菌感染模型获得的，称为人类结肠， 结肠上皮培养物，其来源于从健康供体的活组织检查中分离的成体干细胞。后 在分化过程中，人类结肠产生一层厚的粘液，类似于正常存在于结肠中的粘液。 人结肠。肠出血性大肠杆菌感染导致粘液层的破坏，使肠出血性大肠杆菌能够进入粘膜。 结肠细胞表面这些数据表明，人类类结肠重现了EHEC感染，并提供了独特的 与其他在人类结肠癌细胞系中进行的研究不同的发病机制的见解， 提高对毒力因子作用的认识和对新治疗靶点的评估。我们 假设肠出血性大肠杆菌与结肠粘液相互作用是大肠杆菌初始定植的必要步骤， 上皮导致Stx产生和肠道疾病的全面表现。为了验证这个假设，我们 我建议使用两种互补的肠出血性大肠杆菌感染模型：人结肠和兔，这是 最好的动物模型，可以重现EHEC诱导的人类肠道病变，包括出血性 结肠炎，以达到以下目的：1。确定EHEC的粘液降解是否是必要的， 寄生在人体上皮细胞上2.确定肠出血性大肠杆菌鞭毛与粘蛋白之间是否存在直接相互作用 为EHEC-结肠细胞附着提供初始锚。3.确定肠出血性大肠杆菌与 粘液影响肠出血性大肠杆菌主要毒力因子Stx 2a的产生和释放。结果从 这些实验将进一步阐明肠出血性大肠杆菌与人类相互作用的分子机制。 结肠上皮，特别是粘液层，并确定粘液在EHEC发病机制中的作用。

Summary The proposed work will advance understanding of enteric disease caused by food-borne, Shiga-toxin- producing enterohemorrhagic E. coli (EHEC) constitute the major US cause of life-threatening hemorrhagic colitis and hemolytic uremic syndrome (HUS). While shiga toxin 2a provides 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. We thus focus on early EHEC-host interactions, especially the complex interactions between EHEC and intestinal mucus. Intestinal mucus represents the first line of defense against enteric pathogens and is the first site for the interaction of EHEC with the human colonic epithelium. Our exciting preliminary data suggest that mucus play an important role in EHEC colonization of the human colon. These data were obtained using a novel model of EHEC infection, termed human colonoids, which are primary colonic epithelial cultures derived from adult stem cells isolated from biopsies of healthy donors. Upon differentiation, human colonoids produce a thick layer of mucus similar to that which is normally present in the human colon. EHEC infection results in the destruction of the mucus layer allowing EHEC to gain access to the colonocyte surface. 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. We hypothesize that EHEC interaction with colonic mucus is a necessary step in initial colonization of colonic epithelium leading to Stx production and full manifestation of intestinal disease. To test this hypothesis, we propose to use two complementary models of EHEC infection: human colonoids and the rabbits, which is the best animal model that can recapitulate EHEC-induced human intestinal pathologies, including hemorrhagic colitis, to achieve the following Aims: 1. Determine whether mucus degradation by EHEC is necessary to colonize the human epithelium. 2. Determine whether direct interaction between EHEC flagella and mucin(s) provides an initial anchor for EHEC-colonocyte attachment. 3. Determine whether the interaction of EHEC with mucus affects the production and release of the major EHEC virulence factor, Stx2a. The results gained from the proposed experiments will further elucidate the molecular mechanisms for EHEC interactions with human colonic epithelium, particularly the mucus layer, and establish the role of mucus in EHEC pathogenesis.