Novel E. Coli 0157:H7 Intestinal Colonization Factors

描述(申请人提供)：肠出血性大肠杆菌O157：H7(EHEC)是出血性结肠炎和溶血性尿毒症综合征(HUS)的重要原因。EHEC在大肠内定植，并分泌一种名为志贺毒素(STX)的强大细胞毒素，这种毒素扰乱蛋白质合成，最终导致HUS特有的肾脏损害。感染通过摄入受EHEC污染的食物或水开始，通常是通过接触牛的粪便开始的，牛粪便是该有机体的主要储存库。疾病所需的极具传染性的剂量约为10-100个活生物体，使传播控制变得复杂。首席研究员实验室的前期工作导致了关键的肠道定植因子Inimin的鉴定，发现了一个编码III型分泌系统的大致病岛，该系统介导了肠道组织病理学的特征附着和消失，鉴定了这种病原菌中称为LER的毒力基因的主要调节因子，并发现该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表征EHEC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。其具体目标是：1)利用微阵列和实时定量聚合酶链式反应研究LER调控基因在模拟人体肠道环境和牛肠道中的体内表达；2)利用体外器官培养(IVOC)研究人类宿主上皮细胞对野生型EHEC和等基因效应突变体感染的转录反应；3)表征尿素酶在致病和环境持久性中的作用；以及4)表征编码III型分泌系统转位的LEE4操纵子的转录。拟议的研究将产生关于疾病发病机制的重要新信息，特别是建立肠道定植的最初步骤，以及对牛体内EHEC维持的机制提供洞察力。

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.