PATHOGENESIS OF ETEC INFECTIONS

建议(摘自申请者的摘要)：产肠毒素大肠杆菌(ETEC)是全球感染性腹泻的主要原因，导致 每年有数十万人死亡。目前还没有获得许可的 预防由这些微生物引起的感染的疫苗。此外，许多 基础临床研究的途径仍未探索。长期的 这些研究的目的是寻找ETEC疫苗的可行候选者 发展采用分子发病机制研究，以更全面地了解毒力的基本决定因素。所有以前的疫苗和发病机制 到目前为止，研究主要集中在染色体外质粒编码的毒力上。 各种因素。这里提出的研究试图利用最近的 猪瘟病毒染色体上致病岛(PAI)的发现 ETEC原型菌株H10407，位于SELC基因的两个位点 之前描述的Pais在其他致病性大肠杆菌中。至少有两个基因 在岛上编码的Tia和DleoA在动物模型中影响毒力 感染并调节一种已知的关键毒力因子的释放， 不耐热毒素(LT)。了解促进出口的事件 而将这种毒素传递到真核细胞上的目标受体可能会 事实证明，这对最终开发有效的疫苗是有价值的。这个 本文提出的研究主要集中在PAI在调节中的作用 LT的释放。概述的实验集中在特定的基因座上 在PAI内，包含编码假定的开放阅读框架(ORF) 具有细菌分泌系统共同模体的蛋白质。这些研究是 旨在确定这些基因在调节合成中的作用 和LT的输出。最后，实验性挑战研究将检验 PAI在人感染ETEC毒力中的作用特定的 目的：1.构建定位于所选基因的框内缺失 派。在这个目标中，重点将放在假定的分泌物中的开放阅读框上。 PAI的要素。由此产生的同源缺失突变体的集合 将在随后的实验中用于它们的表型 人物刻画。2.同基因缺失的表型特征 变种人。将对每个突变体进行多项检测，以研究 缺失对毒素合成、输出和传递的影响 真核靶分子。这些实验将被用来证实 研究人员的假设是PAI的额外基因，它们加在一起 编码一个多组份的膜转运蛋白，都需要调制LT 放手。3.蛋白质的亚细胞定位和特性 由PAI上的毒力基因编码。同时评估每个项目的影响 突变后的毒力和毒素释放，个别蛋白质将被 在分子水平上表征，通过实验来定义它们的 亚蜂窝位置。需要这些研究来验证目前的情况 当LT被释放时，这些蛋白质所起作用的假设模型 ETEC的周质间隙。4.框内缺失突变体的检测 ETEC感染的人体实验挑战模型。要检查 PAI在ETEC H10407毒力中的作用 删除突变将在人类身上进行测试。

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.