Fatty acid related regulation of enteric infectious disease

肠道传染病的脂肪酸相关调节

• 批准号: 8030148
• 负责人: Fredrick Jon Kull
• 金额: $ 23.7万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8030148
• 关键词: 5 year old; Accounting; Affect; Age-Years; Amino Acids; Bacteria; Bacterial Infections; Bile fluid; Binding; Biochemical; Biological Assay; Cessation of life; Characteristics; Charge; Child; Cholera; Cholera Toxin; Code; Codon Nucleotides; Communicable Diseases; Country; DNA; DNA Binding; DNA Binding Domain; Development; Diarrhea; Disease; Dysentery; Elderly; Electrophoretic Mobility Shift Assay; Enteral; Enterobacteriaceae; Escherichia coli; Family member; Fatty Acids; Food; Gene Expression; Gene Expression Regulation; Genes; Genetic; Homology Modeling; Individual; Infection; Ingestion; Laboratories; LacZ Genes; Lead; Ligand Binding; Ligands; Lysine; Measures; Mediating; Methods; Modeling; Molecular Conformation; Mutate; Organism; Pathology; Pilum; Play; Positioning Attribute; Process; Production; Proteins; Regulation; Reporter; Resolution; Role; Salmonella; Salmonella enteritidis; Salmonella typhi; Salmonella typhimurium; Sequence Alignment; Shigella flexneri; Side; Signal Transduction; Site-Directed Mutagenesis; Structure; System; Techniques; Testing; Therapeutic; Toxin; Traveler' s diarrhea; Typhoid Fever; United States; Vibrio cholerae; Virulence; Virulence Factors; Water; World Health Organization; Yersinia enterocolitica; base; enteropathogenic Escherichia coli; enterotoxigenic Escherichia coli; gel mobility shift assay; interest; member; palmitoleic acid; pathogenic bacteria; prevent; promoter; prophylactic; response; therapeutic development; transcription factor

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.

描述（由申请人提供）：据世界卫生组织称，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，其中许多是五岁以下儿童。在美国，腹泻是第二常见的传染病，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。一旦被宿主摄入，肠道中的环境信号就会启动毒力基因级联反应，导致毒力因子的产生。这些毒力因子（其中许多是毒素、效应分子和定植因子）的表达会导致多种使人衰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，主要毒力因子是毒素共调节菌毛（TCP）和霍乱毒素（CT），前者是生物体在宿主中定植所必需的，后者是导致疾病病理的原因。两种毒力因子的表达均受 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 AraC/XylS (A/X) 调节因子超家族的成员，该家族在细菌世界中有众多成员，可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域和 DNA 结合域，将其锁定在非活性构象。两个带正电荷的赖氨酸侧链（每个结构域各有一个）在脂肪酸结合中发挥着关键作用，从而在毒力基因调控机制中发挥着关键作用。 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（如果不相同）的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 蛋白超家族的候选者，这些候选者在与 ToxT 中发现的氨基酸残基同源的位置上含有带正电荷的氨基酸残基。脂肪酸对各种候选生物体中毒力基因产生的影响将利用 lacZ 融合系统进行测量，并利用电泳迁移率变动测定 (EMSA)、基于 NMR 的配体结合测定和定点诱变进行确认。初步分析表明，许多病原菌，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氏菌，都含有具有同源带正电残基和配体结合袋的A/X调节因子。这里提出的研究将证实这些细菌是否使用常见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引起旅行者腹泻、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：肠道感染性腹泻病每年影响全世界超过 40 亿人，估计导致 400 万至 600 万人死亡，其中主要是老年人和 5 岁以下儿童。该提案研究了病原菌如何利用脂肪酸来调节毒力活性。了解这一过程可能会导致治疗或预防肠道细菌感染（包括旅行者腹泻、沙门氏菌、痢疾和伤寒）的疗法的开发。