Functions of Translocated Bacterial Glycosyltransferases

易位细菌糖基转移酶的功能

• 批准号: 9222103
• 负责人: Philip Ross Hardwidge
• 金额: $ 18.75万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-07 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9222103
• 关键词: Arginine; Bacterial Proteins; Binding; Carbohydrates; Citrobacter rodentium; Data; Development; Diarrhea; Enzymatic Biochemistry; Enzymes; Epithelial Cells; Escherichia coli; Escherichia coli EHEC; Escherichia coli Infections; Event; Family; Food; Future; Gastroenteritis; Glycolysis; Goals; Health; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; Human; Immune system; Infection; Inflammatory; Injectable; Innate Immune Response; Intestines; Investigation; Kidney Failure; Knowledge; Link; Modification; Molecular; Mus; Names; Natural Immunity; Orthologous Gene; Pathway interactions; Pharmacology; Phosphorylation; Post-Translational Protein Processing; Property; Proteins; Publishing; Reporting; Research; Role; Salmonella enterica; Salmonella typhimurium; Serine; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Source; Substrate Specificity; TNF Receptor-Associated Factors; Threonine; Toxin; Tumor Necrosis Factor Receptor; Type III Secretion System Pathway; Virulence; Virulence Factors; Water; design; enteropathogenic Escherichia coli; glycosyltransferase; improved; in vivo; innate immune function; innovation; interdisciplinary approach; pathogen; prevent; protein function; response; small molecule

Project Summary. Infectious diarrhea constitutes a major endemic health threat as an increasingly frequent and deadly source of food- and water-borne illness. Enterohemorrhagic Escherichia coli (EHEC) are especially significant bacterial pathogens because their toxins can cause a type of renal failure (hemolytic uremic syndrome) for which therapy is limited. The molecular mechanisms by which EHEC and the closely related enteropathogenic E. coli (EPEC), as well as Salmonella enterica serovar Typhimurium, a common cause of human gastroenteritis, inhibit host innate immune responses to promote bacterial colonization are under intense investigation. Understanding these host-pathogen dynamics in molecular detail may enhance the development of pharmacological approaches to prevent and treat infections. Through studies of the mechanism of the NleB virulence protein expressed by E. coli and by a related pathogen of mice, Citrobacter rodentium, it was determined that NleB functions as a glycosyltransferase enzyme that covalently modifies host proteins with N-acetylglucosamine (GlcNAc) to subvert their normal functions. NleB disrupts tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) signaling, leading to inhibition of the pro-inflammatory NF-κB pathway. The glycolysis enzyme GAPDH functions as a co- activator of TRAF2 activity. The modification of GAPDH with GlcNAc by NleB prevents GAPDH from binding to and activating TRAF2, leading to a reduced NF-κB response to infection and thus enhanced bacterial survival. It was also discovered that some NleB orthologs modify the arginine residues of host proteins, rather than the expected modification of their serine/threonine residues. S. Typhimurium strains also encode from 1-3 NleB orthologs named SseK/1/2/3, although their enzymatic activities and relative contributions to virulence are less clear. The proposed project is designed to elucidate the molecular mechanisms of NleB/SseK glycosyltransferase activities, with the ultimate goal of using these data to inform future strategies to prevent and treat infectious diarrhea. The specific aims are: 1) Elucidate the NleB/SseK GlcNAcylation mechanisms and their targeting of host proteins. 2) Identify GAPDH GlcNAcylation sites and characterize their role in GAPDH activity and NF-κB pathway activation. This multidisciplinary approach provides an opportunity to probe not only the enzymology of these virulence factors, but also to effect the future development of small molecules with anti-virulence properties.

项目摘要。 感染性腹泻是一种主要的地方性健康威胁，是一种日益频繁和致命的传染源， 食源性和水源性疾病。肠出血性大肠杆菌（EHEC）是一种重要的细菌， 病原体，因为它们的毒素可导致一种类型的肾衰竭（溶血性尿毒综合征）， 治疗有限。EHEC及其密切相关的肠致病性E.杆菌 （EPEC），以及肠道沙门氏菌血清型鼠伤寒，一种人类胃肠炎的常见原因， 抑制宿主先天性免疫应答以促进细菌定殖正在进行深入研究。 从分子水平上了解这些宿主-病原体动力学， 预防和治疗感染的药理学方法。 通过对E.大肠杆菌和相关的 小鼠的病原体，啮齿柠檬酸杆菌，确定NleB作为糖基转移酶起作用 用N-乙酰葡糖胺（GlcNAc）共价修饰宿主蛋白质以破坏其正常 功能协调发展的NleB破坏肿瘤坏死因子受体（TNFR）相关因子2（TRAF 2）信号传导，导致 抑制促炎性NF-κB通路。糖酵解酶GAPDH的功能是作为一种辅助酶， TRAF 2活性的激活剂。通过NleB用GlcNAc修饰GAPDH阻止GAPDH结合至NleB。 并激活TRAF 2，导致NF-κB对感染的反应降低，从而提高细菌存活率。 还发现，一些NleB直向同源物修饰宿主蛋白的精氨酸残基，而不是宿主蛋白的精氨酸残基。 它们的丝氨酸/苏氨酸残基的预期修饰。S.鼠伤寒菌株也编码1-3个NleB 命名为SseK/1/2/3的直系同源物，尽管它们的酶活性和对毒力的相对贡献较小 清楚 该项目旨在阐明NleB/SseK的分子机制 糖基转移酶活性，最终目标是利用这些数据为未来的预防策略提供信息， 并治疗感染性腹泻。具体目的是：1）阐明NleB/SseK GlcNAc酰化机制 以及它们对宿主蛋白质的靶向作用。2)鉴定GAPDH GlcNAc化位点并表征其在 GAPDH活性和NF-κB通路活化。这种多学科方法提供了一个机会， 不仅探讨这些毒力因子的酶学，而且对未来小分子生物学的发展也有一定的影响。 具有抗毒性特性的分子。