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

项目 3 - 人类结肠作为肠出血性大肠杆菌病理学模型

• 批准号: 9150902
• 负责人: JAMES B KAPER
• 金额: $ 28.95万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9150902
• 关键词: Address; Adherent Culture; Adopted; Animal Model; Antibiotics; Apical; Bacterial Attachment Site; Biopsy; Cell Line; Cells; Cessation of life; Characteristics; Chloride Ion; Chlorides; Clinical Data; Coculture Techniques; Colon; Complex; Data; Development; Diarrhea; Disease; Enteral; Enteroendocrine Cell; Epithelial; Epithelial Cells; Epithelium; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; Etiology; Face; Goblet Cells; Grant; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; Hospitalization; Human; Immune; Immune response; Infection; Intestinal Diseases; Intestines; Ion Transport; Ions; Life; Medical; Modeling; Molecular; Molecular Profiling; Mucous body substance; Organoids; Pathogenesis; Patients; Play; Production; Risk; Rodent Model; Role; Septic Toxemia; Serine Protease; Shiga Toxin; Shigella Infections; Surface; System; Systemic disease; Technology; Testing; Thick; Virulence; Virulence Factors; absorption; abstracting; adult stem cell; base; cellular microvillus; colon cancer cell line; cytokine; effective therapy; foodborne; human disease; human stem cells; human subject; improved; individual patient; insight; intestinal epithelium; macrophage; microbial; monolayer; neutrophil; new therapeutic target; novel; novel therapeutic intervention; pathogen; research study; stem cell biology

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.

摘要 拟议的研究将促进对食源性志贺毒素引起的肠道疾病的理解， 产生肠出血性E.大肠埃希氏菌（EHEC）是导致危及生命的出血性结肠炎的主要原因 和溶血性尿毒综合征而滋贺毒素2a是肠源性和 肠外疾病的表现，临床数据强烈表明，Stx-毒血症发生在疾病的早期 并且是短暂的。因此，我们的研究将集中在早期EHEC-宿主相互作用，特别是复合物 肠出血性大肠杆菌与人结肠细胞、杯状细胞和免疫细胞（包括巨噬细胞）的相互作用 和中性粒细胞。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， 人结肠样单层（HCM），其是来源于成体干细胞的原代结肠上皮培养物 分离自健康供体的结肠活组织检查。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， 产生一层厚的粘液，类似于人类结肠中正常存在的粘液。使用HCM 我们在这里证明，肠出血性大肠杆菌感染导致粘液层破坏，使肠出血性大肠杆菌 进入结肠细胞表面。丝氨酸蛋白酶EspP在肠出血性大肠杆菌定植中起作用， 诱导离子分泌，表明其在EHEC诱导的水样腹泻中的作用。这些数据表明，人类 类结肠概括了肠出血性大肠杆菌感染，并提供了独特的见解，发病机制不同于其他 在人类结肠癌细胞系中进行的研究，使人们能够更好地了解毒力的作用， 因素和评估新的治疗目标。与此病原体相关，我们将测试假设， HCM是唯一代表离体人结肠上皮细胞的，它重现了人EHEC感染， 在发病机理上提供了独特的见解。为了验证这一假设并加深我们对肠出血性大肠杆菌的了解 肠道发病机制，我们提出以下目的：1。确定丝氨酸蛋白酶EspP在 肠出血性大肠杆菌在人结肠上皮的定植和毒力的发展。2.测定分子 EHEC引起水样腹泻的机制。3.确定巨噬细胞和中性粒细胞在 EHEC诱导的免疫应答。从拟议的实验中获得的结果将进一步阐明 EHEC与人类结肠上皮相互作用的分子机制，测试EspP丝氨酸的作用 蛋白酶在肠出血性大肠杆菌定植和水样腹泻发展中的作用以及中性粒细胞 和巨噬细胞清除肠出血性大肠杆菌。