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

摘要 拟议的工作将促进对由食源性志贺毒素引起的肠道疾病的了解。 产肠出血性大肠杆菌(EHEC)是美国危及生命的出血性疾病的主要原因 结肠炎和溶血性尿毒症综合征(HUS)。而志贺毒素2a提供了主要的毒力因子 肠道和肠外疾病的表现，临床资料强烈提示STX-中毒血症的发生 发病早，寿命短。因此，我们关注早期EHEC与宿主的相互作用，特别是复合体 肠出血性大肠杆菌与肠道粘液的相互作用。肠道粘液是抵抗病毒的第一道防线 肠道病原体，是EHEC与人结肠上皮相互作用的第一个部位。我们的 令人振奋的初步数据表明，粘液在EHEC在人类结肠的定植中起着重要作用。 这些数据是通过一种新的EHEC感染模型获得的，这种模型被称为人类结肠炎，这是主要的 从健康捐献者的活检组织中分离的成人干细胞来源的结肠上皮细胞培养。vt.在.的基础上 在分化过程中，人类结肠腺会产生一层厚厚的粘液层，类似于正常情况下存在于 人的结肠。EHEC感染导致粘液层的破坏，使EHEC能够接触到 结肠细胞表面。这些数据表明，人类类结肠杆菌重现了EHEC感染，并提供了独特的 对不同于在人类结肠癌细胞系中进行的其他研究的发病机制的见解，允许 提高了对毒力因子作用的认识和对新的治疗靶点的评估。我们 假设EHEC与结肠粘液的相互作用是结肠初始定植的必要步骤 导致STX产生的上皮细胞和肠道疾病的充分表现。为了检验这一假设，我们 建议使用两种相辅相成的EHEC感染模型：人类结肠炎和兔，这是 能概括EHEC诱导的包括出血在内的人类肠道病理的最佳动物模型 结肠炎，以实现以下目标：1.确定EHEC是否有必要降解粘液以 在人类上皮细胞上定植。2.确定肠出血性大肠杆菌鞭毛与粘蛋白是否直接相互作用(S) 为EHEC-结肠癌细胞附着提供初始锚。3.确定EHEC与HEC的相互作用 粘液影响EHEC主要毒力因子Stx2a的产生和释放。从以下方面获得的结果： 拟议的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.