Novel E. Coli 0157:H7 Intestinal Colonization Factors

描述（由申请方提供）：肠出血性大肠杆菌O 157：H7（EHEC）是出血性结肠炎和溶血性尿毒综合征（HUS）的重要病因。肠出血性大肠杆菌在大肠内定植，并分泌一种称为滋贺毒素（Stx）的强效细胞毒素，该毒素破坏蛋白质合成，最终导致HUS特征性肾损伤。感染始于摄入被肠出血性大肠杆菌污染的食物或水，通常是由于接触牛粪便（该生物的主要宿主）。疾病所需的极高传染性剂量，约。10 - 100个活微生物，使传播控制复杂化。在主要研究者实验室的先前工作导致了关键的肠道定植因子的鉴定，称为内膜，发现了编码III型分泌系统的大致病岛，该分泌系统介导了特征性的附着和消退肠道组织病理学，描述了这种病原体中毒力基因的主要调节因子，称为Ler，以及发现这种病原体中的关键毒力因子受群体感应调节。 下一个支持期的拟议研究目标包括继续描述肠出血性大肠杆菌的致病机制，以及旨在确定在牛宿主中维持这种病原体的关键因素的研究。本研究的具体目的是：1）利用微阵列和定量真实的时间PCR技术，研究Ler调节子的特性，并评估Ler调节基因在模拟人肠道环境和牛肠道中的体内表达; 2）利用体外器官培养（IVOC）技术，研究人宿主上皮细胞对野生型EHEC和同基因效应突变体感染的转录反应; 3）表征尿素酶在发病机理和环境持久性中的作用;和4）表征LEE 4操纵子的转录，其编码III型分泌系统易位子。拟议的研究应产生重要的新信息，疾病的发病机制，特别是在建立肠道定植的初始步骤，以及提供见解的机制，负责维护肠出血性大肠杆菌在牛水库。

DESCRIPTION (provided by applicant): Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important cause of hemorrhagic colitis and the hemolytic uremic syndrome (HUS). EHEC colonizes the large intestine and secretes a potent cytotoxin called Shiga toxin (Stx) that disrupts protein synthesis eventually leading to kidney damage characteristic of HUS. Infection begins by ingestion of food or water that is contaminated with EHEC, often from contact with cattle feces, the primary reservoir of the organism. The extremely infectious dose required for disease, ca. 10 - 100 viable organisms, complicates control of transmission. Previous work in the principal investigator's laboratory resulted in the identification of the crucial intestinal colonization factor called intimin, the discovery of a large pathogenicity island encoding a type III secretion system that mediates the characteristic attaching and effacing intestinal histopathology, the characterization of a master regulator of virulence genes in this pathogen called Ler, and the discovery that key virulence factors in this pathogen are regulated by quorum sensing. The proposed research goals for the next period of support include continued characterization of the pathogenic mechanisms of EHEC as well as studies aimed at identifying key factors that are involved in maintenance of this pathogen in the bovine reservoir. The specific aims are 1) Characterize the Ler regulon and assess in vivo expression of Ler-regulated genes in a simulated human intestinal environment and bovine intestinal tract using microarrays and quantitative real time PCR; 2) Characterize human host epithelial cell transcriptional responses to infection with wild type EHEC and isogenic effector mutants using in vitro organ culture (IVOC); 3) Characterize the role of urease in pathogenesis and environmental persistence; and 4) Characterize transcriptional of the LEE4 operon, which encodes the the type III secretion system translocon. The proposed studies should yield significant new information regarding the pathogenesis of disease, particularly the initial steps in establishing intestinal colonization, as well as provide insights into the mechanisms responsible for maintenance of EHEC in the bovine reservoir.