Fatty acid related regulation of enteric infectious disease

描述（由申请人提供）：据世界卫生组织称，全世界每年约有40亿人因肠道感染而腹泻，估计造成400万至600万人死亡，其中许多是五岁以下儿童。在美国，腹泻是第二大最常见的传染病，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，肠道疾病是由通过受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。这些毒力因子的表达，其中许多是毒素、效应分子和定植因子，导致各种各样的使人虚弱且往往致命的腹泻疾病，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协同调节菌毛（TCP）和霍乱毒素（CT），前者是生物体在宿主中定殖所必需的，后者是造成霍乱病理的原因。这两种毒力因子的表达均受弓形虫的调控，弓形虫是霍乱弧菌毒力基因表达的主要调控因子。ToxT是AraC/XylS （a /X）调节超家族的一员，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控域和DNA结合域，将其锁定在非活性构象中。两个带正电的赖氨酸侧链，分别来自两个结构域，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。

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.