Fatty acid related regulation of enteric infectious disease

描述(申请人提供)：根据世界卫生组织的数据，由肠道感染引起的腹泻每年影响全球约40亿人，估计导致400万至600万人死亡，其中许多是5岁以下的儿童。在美国，腹泻是第二种最常见的传染病，在一些国家，腹泻疾病占所有死亡人数的三分之一。在许多情况下，肠道疾病是由通过受污染的食物或水摄入致病菌引起的。一旦被宿主摄取，肠道中的环境信号就会启动毒力基因级联，导致毒力因子的产生。这些毒力因子的表达，其中许多是毒素、效应分子和定居因子，导致各种衰弱的、通常是致命的腹泻疾病，包括旅行者的腹泻、痢疾和霍乱。在霍乱中，主要的毒力因子是毒素共调节菌毛(TCP)和霍乱毒素(CT)，前者是机体定植宿主所必需的，后者是疾病的病理基础。这两种毒力因子的表达都受霍乱弧菌毒力基因表达的最高调节因子ToxT的调控。ToxT是AraC/XylS(A/X)超家族的成员之一，在细菌世界中有许多成员，它被胆汁中存在的脂肪酸抑制。在ToxT的结构中，棕榈油酸连接转录因子的调节域和DNA结合域，将其锁定在不活跃的构象中。两个带正电的赖氨酸侧链，每个结构域一个，在脂肪酸结合中发挥关键作用，从而在毒力基因调控机制中发挥关键作用。 这项申请中提出的研究将检验这样一个假设，即许多其他肠道细菌使用类似的(如果不是完全相同的)机制来调节致病活动。该方法将利用二级结构预测和同源建模来从A/X蛋白超家族中鉴定候选基因，该家族含有与ToxT中发现的氨基酸同源的正电荷氨基酸残基。脂肪酸对各种候选生物毒力基因产生的影响将使用LacZ融合系统进行测量，并使用凝胶迁移率改变分析(EMSA)、基于核磁共振的配体结合分析和定点突变来证实。初步分析表明，一些病原菌，包括几种类型的大肠杆菌、伤寒沙门氏菌、鼠伤寒沙门氏菌、福氏志贺氏菌和小肠结肠炎耶尔森菌，都含有A/X调节剂，带有同源的正电荷残基和配体结合口袋。这里提出的研究将证实这些细菌是否使用一种常见的脂肪酸介导的机制来调节致病活动，从而为开发可广泛应用于治疗导致旅行者腹泻、痢疾和伤寒等疾病的肠道细菌感染的疗法奠定基础。 公共卫生相关性：全世界每年有40多亿人感染肠道感染性腹泻疾病，估计有400万至600万人死亡，主要是老年人和5岁以下儿童。这项建议调查了病原菌如何使用脂肪酸来调节毒力活动。了解这一过程可能会导致开发治疗或预防肠道细菌感染的疗法，包括旅行者的腹泻、沙门氏菌、痢疾和伤寒。

DESCRIPTION (provided by applicant): According to the World Health Organization, diarrhea caused by enteric infections affects some four billion individuals annually throughout the world, resulting in an estimated four million to six million deaths, many in children under five years of age. In the United States, diarrhea is the second most common infectious illness, and in some countries diarrheal diseases account for up to one third of all deaths. In many cases, enteric disease is caused by the ingestion of pathogenic bacteria via contaminated food or water. Once ingested by the host, environmental signals in the gut initiate a virulence gene cascade leading to the production of virulence factors. The expression of these virulence factors, many of which are toxins, effector molecules, and colonization factors, results in a wide variety of debilitating and often deadly diarrheal diseases including travelers' diarrhea, dysentery, and cholera. In cholera the primary virulence factors are the toxin- coregulated pilus (TCP), which is required for the organism to colonize the host, and cholera toxin (CT), which is responsible for the pathology of the disease. The expression of both virulence factors is regulated by ToxT, the paramount regulator of virulence gene expression in Vibrio cholerae. ToxT, a member of the AraC/XylS (A/X) superfamily of regulators, of which there are numerous members throughout the bacterial world, is inhibited by fatty acids present in bile. In the structure of ToxT, a palmitoleic acid bridges the regulatory and DNA binding domains of the transcription factor, locking it in an inactive conformation. Two positively charged lysine side chains, one from each domain, play a critical role in fatty acid binding and, thereby, in the mechanism of virulence gene regulation. The studies proposed in this application will test the hypothesis that many other enteric bacteria use a similar, if not identical, mechanism to regulate pathogenic activity. The approach will utilize secondary structure prediction and homology modeling to identify candidates from the A/X protein superfamily containing positively charged amino acid residues at positions homologous to those found in ToxT. The effect of fatty acids on virulence gene production in the various candidate organisms will be measured utilizing a lacZ fusion system and confirmed using electrophoretic mobility shift assays (EMSAs), an NMR-based ligand binding assay, and site directed mutagenesis. Preliminary analysis has indicated that a number of pathogenic bacteria, including several types of Escherichia coli, Salmonella typhi, Salmonella typhimurium, Shigella flexneri, and Yersinia enterocolitica, contain A/X regulators with homologous positively charged residues and ligand binding pockets. The studies proposed here will confirm if these bacteria use a common, fatty acid mediated mechanism to regulate pathogenic activity, thereby laying the groundwork for development of therapeutics that could be broadly applied to treat enteric bacterial infections causing diseases such as travelers' diarrhea, dysentery, and typhoid fever. PUBLIC HEALTH RELEVANCE: Enteric infectious diarrheal diseases affect more than 4 billion individuals yearly throughout the world, resulting in an estimated 4 million to 6 million deaths, primarily among the elderly and children less than 5 years of age. This proposal investigates how pathogenic bacteria use fatty acids to regulate virulence activity. Understanding this process could lead to the development of therapeutics to treat or prevent enteric bacterial infections including travelers' diarrhea, salmonella, dysentery, and typhoid fever.