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

粘液在肠出血性大肠杆菌定植于人类结肠和肠道疾病发展中的作用

• 批准号: 9351785
• 负责人: OLGA KOVBASNJUK
• 金额: $ 14.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9351785
• 关键词: Adherent Culture; Adopted; Affect; Animal Model; Antibiotics; Apical; Bacteria; Binding; Biological Assay; Biopsy; Cell Communication; Cell Line; Cessation of life; Cleaved cell; Clinical Data; Colon; Complex; Data; Development; Diarrhea; Disease; Distal; Energy-Generating Resources; Enteral; Enzymes; Epithelial; Epithelial Attachment; Epithelial Cells; Epithelium; Escherichia coli EHEC; Escherichia coli Infections; Face; Flagella; Goals; Grant; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; Hospitalization; Human; In Vitro; Infection; Intestinal Diseases; Intestines; Life; Mediating; Medical; Modeling; Molecular; Mucin-2 Staining Method; Mucins; Mucous body substance; Oryctolagus cuniculus; Pathogenesis; Pathology; Patients; Phase; Play; Production; Risk; Role; Septic Toxemia; Shiga Toxin; Site; Staging; Stem cells; Surface; Systemic disease; Technology; Testing; Thick; Time; Toxin; Virulence; Virulence Factors; Work; adult stem cell; base; colon cancer cell line; effective therapy; enteric pathogen; foodborne; human stem cells; human subject; improved; individual patient; insight; intestinal epithelium; microbial; monolayer; mutant; new therapeutic target; novel; novel therapeutic intervention; research study; stem cell biology; sugar; therapeutic target; uptake

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.

概括 拟议的工作将提高人们对食物传播，shiga-toxin-引起的肠道疾病的理解。 产生肠肠肠肠肠球大肠杆菌（EHEC）构成了美国危及生命的出血的主要原因 结肠炎和溶血性尿毒症综合征（HUS）。虽然Shiga Toxin 2a提供了主要的毒力因子 肠道和肠外疾病表现，临床数据强烈表明发生了STX毒血症 在疾病的早期，是短暂的。因此，我们专注于早期的EHEC-HOST互动，尤其是复杂的 EHEC和肠粘膜之间的相互作用。肠道粘液代表了针对的第一道防线 肠道病原体，是EHEC与人类结肠上皮相互作用的第一个部位。我们的 令人兴奋的初步数据表明，粘液在人类结肠的EHEC定植中起重要作用。 这些数据是使用新型EHEC感染模型获得的，称为人类结构菌，这是主要的 从健康供体的活检中分离出的成年干细胞的结肠上皮培养物。之上 分化，人类结肠造成的粘液层类似于通常存在于 人类结肠。 EHEC感染导致粘液层破坏，使EHEC能够进入 结肠细胞表面。这些数据表明，人类结肠局部概括EHEC感染并提供独特 对发病机理的见解，与在人类结肠癌细胞系中进行的其他研究不同，允许 改善了对毒力因子的作用以及对新型治疗靶标的评估的认识。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 上皮导致STX产生和肠道疾病的完全表现。为了检验这一假设，我们 建议使用两个EHEC感染的互补模型：人类结肠和兔子，这是 最佳的动物模型可以概括EHEC诱导的人肠道病理，包括出血 结肠炎，以达到以下目的：1。确定EHEC是否需要粘液降解 殖民人类上皮。 2。确定EHEC鞭毛和粘蛋白之间的直接相互作用是否直接相互作用 为EHEC - 核细胞附着提供了初始锚。 3。确定EHEC的相互作用是否与 粘液会影响主要EHEC毒力因子STX2A的产生和释放。从中得出的结果 提出的实验将进一步阐明EHEC与人相互作用的分子机制 结肠上皮，尤其是粘液层，并确定粘液在EHEC发病机理中的作用。