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，因为它是一种日益频繁和致命的 通过食物和水传播的疾病。肠出血性大肠杆菌(EHEC)是一种特别重要的细菌 病原体，因为它们的毒素可以导致一种类型的肾功能衰竭(溶血性尿毒症综合征) 治疗是有限的。肠出血性大肠杆菌与致病性肠出血性大肠杆菌分子机制的研究进展 (EPEC)，以及人类胃肠炎的常见原因--鼠伤寒沙门氏菌， 抑制宿主先天免疫反应以促进细菌定植的研究正在紧张进行中。 在分子细节上了解这些寄主-病原体的动力学可能会促进 预防和治疗感染的药理学方法。 通过对大肠杆菌和相关菌株表达的NleB毒力蛋白机制的研究 小鼠的病原体轮状柠檬酸杆菌，已确定NleB具有糖基转移酶的功能 与N-乙酰氨基葡萄糖(GlcNAc)共价修饰宿主蛋白以颠覆其正常状态的酶 功能。NleB干扰肿瘤坏死因子受体(TNFR)相关因子2(TRAF2)信号，导致 抑制促炎因子-κB途径。糖酵解酶GAPDH的功能是辅酶-2。 TRAF2活性激活剂。NleB用GlcNAc修饰GAPDH可阻止GAPDH与 激活TRAF2，导致对感染的NF-κB反应减少，从而提高细菌存活率。 还发现一些NleB同源基因可以修饰宿主蛋白的精氨酸残基，而不是 预期对其丝氨酸/苏氨酸残基进行修饰。鼠伤寒沙门氏菌也编码1-3个NleB 同源基因命名为SseK/1/2/3，尽管它们的酶活性和对毒力的相对贡献较小 安全。 该项目旨在阐明NleB/SseK的分子机制 糖基转移酶活动，最终目的是利用这些数据为未来的预防战略提供信息 治疗感染性腹泻。具体目的是：1)阐明NleB/SseK GlcN酰化机制 以及它们对宿主蛋白的靶向。2)确定GAPDHGlcN酰化位点并表征它们在 GAPDH活性和NF-κB通路的激活。这种多学科的方法提供了一个机会 不仅探讨了这些毒力因子的酶学性质，而且还影响了小球虫的未来发展。 具有抗病毒特性的分子。