Fatty acid related regulation of enteric infectious disease

描述（由申请人提供）：根据世界卫生组织的数据，肠道感染引起的腹泻每年影响全世界约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，腹泻是第二常见的传染病，在一些国家，腹泻病占所有死亡人数的三分之一。在许多情况下，肠道疾病是由通过污染的食物或水摄入致病菌引起的。一旦被宿主摄入，肠道中的环境信号启动毒力基因级联，导致毒力因子的产生。这些毒力因子（其中许多是毒素、效应分子和定殖因子）的表达导致多种使人衰弱且通常致命的肠道疾病，包括旅行者腹泻、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），毒素共调节菌毛（TCP）是生物体定殖宿主所需的，霍乱毒素（CT）是疾病的病理学原因。这两种毒力因子的表达受ToxT调节，ToxT是霍乱弧菌毒力基因表达的最重要调节因子。ToxT是调节因子AraC/XylS（A/X）超家族的成员，该家族在整个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，棕榈油酸桥接转录因子的调节和DNA结合结构域，将其锁定在非活性构象中。两个带正电荷的赖氨酸侧链，每个域一个，在脂肪酸结合中起关键作用，从而在毒力基因调控机制中起关键作用。 本申请中提出的研究将检验以下假设：许多其他肠道细菌使用类似的（如果不相同的话）机制来调节致病活性。该方法将利用二级结构预测和同源性建模来识别来自A/X蛋白超家族的候选物，所述A/X蛋白超家族在与ToxT中发现的那些同源的位置处含有带正电荷的氨基酸残基。将使用lacZ融合系统测量脂肪酸对各种候选微生物中毒力基因产生的影响，并使用电泳迁移率变动试验（EMSA）、基于NMR的配体结合试验和定点诱变进行确认。初步分析表明，许多致病菌，包括几种类型的大肠杆菌，伤寒沙门氏菌，鼠伤寒沙门氏菌，福氏志贺菌和小肠结肠炎耶尔森氏菌，含有A/X调节剂与同源的带正电荷的残基和配体结合口袋。本文提出的研究将证实这些细菌是否使用共同的脂肪酸介导的机制来调节致病活性，从而为开发可广泛应用于治疗肠道细菌感染引起的疾病（如旅行者腹泻，痢疾和伤寒）的治疗方法奠定基础。

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.