PATHOGENESIS OF ETEC INFECTIONS

提案（改编自申请人摘要）：产肠杆菌性大肠杆菌 大肠杆菌（ETEC）是全球感染性腹泻的主要原因， 每年有成千上万的人死亡。 目前没有许可证 疫苗，以防止由这些生物体引起的感染。 而且很多 基础临床研究的途径仍未探索。 长期 这些研究的目的是发现ETEC疫苗的可行候选物 发展采用分子发病机制的研究，以更充分地了解 毒力的基本决定因素。 所有既往疫苗和发病机制 迄今为止的研究主要集中在染色体外（质粒）编码的毒力 因素 这里提出的研究试图利用最近的 发现了一个致病岛（派）在染色体的 原型ETEC菌株H10407，位于selC基因的两个位点， 先前描述的PAI在其他致病性E.杆菌 至少两种基因 在岛上编码的tia和DleoA，影响了动物模型的毒力。 感染并调节已知的关键毒力因子的释放， 不耐热毒素（LT）。 了解有助于导出的事件 将这种毒素传递到真核细胞上的靶受体， 在最终研制出有效的疫苗方面证明是有价值的。 的 本文提出的研究主要集中在派在调节 LT的释放。概述的实验集中在一个特定的位点 在派中，含有编码推定的 具有细菌分泌系统共有基序的蛋白质。 这些研究 旨在确定这些基因在调节合成中的作用， 和LT的输出。最后，实验挑战研究将检查 派对人感染ETEC毒力的贡献。 具体 目的：1. 在位于基因组DNA上的选定基因中构建框内缺失。 派代 在这个目标中，重点将放在推定的分泌物中的ORF上 派的元素。 由此产生的同基因缺失突变体的集合 然后将用于随后针对其表型的实验 特征化 2. 同基因缺失的表型表征 变种人 每种突变体将在多项试验中进行检查， 缺失对毒素合成、输出和递送至 真核生物靶分子。 这些实验将用于确认 研究人员假设派的其他基因共同作用 编码多组分膜转运蛋白，是调节LT所必需的 release. 3. 蛋白质的亚细胞定位和表征 由派上的毒力基因编码。 在评估每种影响的同时 突变的毒力和毒素释放，个别蛋白质将 在分子水平上进行了表征， 亚细胞定位 这些研究将需要验证目前的 这些蛋白质在LT释放时所起作用的假设模型 ETEC的周质空间。 4.在一个实施例中测试框内缺失突变体， ETEC感染人类实验攻击模型。 审查 派对ETEC H10407毒力的贡献，DleoA框内 将在人类中测试缺失突变体。

PROPOSAL (Adapted from the applicant's abstract): Enterotoxigenic Escherichia coli (ETEC) are a leading cause of infectious diarrhea worldwide, causing hundreds of thousands of deaths each year. There is presently no licensed vaccine to prevent infections caused by these organisms. Furthermore, many avenues of basic clinical investigation remain unexplored. The long-term objective of these studies is to discover viable candidates for ETEC vaccine development employing molecular pathogenesis studies to more fully understand the essential determinants of virulence. All prior vaccine and pathogenesis studies to date have focused on extrachromosomal (plasmid)-encoded virulence factors. The studies proposed here attempt to capitalize on the recent discovery of a pathogenicity island (PAI) in the chromosome of the prototypical ETEC strain H10407, located in the selC gene, the site of two previously described PAIs in other pathogenic E. coli. At least two genes encoded on the island, tia and DleoA, affect virulence in an animal model of infection and modulate the release of a known critical virulence factor, the heat-labile toxin (LT). Understanding the events which facilitate the export and delivery of this toxin to its target receptors on eukaryotic cells could prove valuable in the ultimate development of an effective vaccine. The studies proposed herein focus primarily on the role of the PAI in modulating the release of LT. The outlined experiments concentrate on a specific locus within the PAI that contains open reading frames (ORFs) encoding putative proteins with motifs common to bacterial secretion systems. These studies are directed at ascertaining the role of these genes in modulating the synthesis and export of LT. Finally, experimental challenge studies will examine the contribution of the PAI to virulence of ETEC in human infection. Specific Aims: 1. Construction of in-frame deletions in selected genes located on the PAI. In this aim, the focus will be on ORFs within a putative secretion element of the PAI. The resulting collection of isogeneic deletion mutants will then be used in subsequent experiments directed at their phenotypic characterization. 2. Phenotypic characterization of isogeneic deletion mutants. Each mutant will be examined in a number of assays to investigate the effects of the deletion on toxin synthesis, export, and delivery to eukaryotic target molecules. These experiments will be used to confirm the investigator's hypothesis that additional genes of the PAI, which together encode a multicomponent membrane transporter, are required to modulate LT release. 3. Subcellular localization and characterization of proteins encoded by virulence genes on the PAI. While assessing the impact of each mutation on virulence and toxin release, the individual proteins will be characterized on a molecular level with experiments to define their subcellular location. These studies will be needed to validate the present hypothetical model of the role played by these proteins as LT is released from the periplasmic space of ETEC. 4. Testing of an in-frame deletion mutant in a human experimental challenge model of ETEC infection. To examine the contribution of the PAI to virulence of ETEC H10407, the DleoA in-frame deletion mutant will be tested in humans.