Functions of Translocated Bacterial Glycosyltransferases

易位细菌糖基转移酶的功能

基本信息

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
项目类别：
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

项目摘要

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。感染性腹泻作为一种日益频繁和致命的传染源，构成了一种主要的地方性健康威胁。肠出血性大肠杆菌 (EHEC) 是特别重要的细菌性疾病。病原体，因为它们的毒素可导致一种肾衰竭（溶血性尿毒症综合征）肠出血性大肠杆菌和密切相关的致病性大肠杆菌的分子机制是有限的。 (EPEC) 以及鼠伤寒沙门氏菌（人类胃肠炎的常见原因），抑制宿主先天免疫反应以促进细菌定植正在深入研究中。了解这些宿主-病原体动态的分子细节可能会促进预防和治疗感染的药理学方法。通过研究大肠杆菌及相关蛋白表达的NleB毒力蛋白的机制小鼠病原体，啮齿类柠檬酸杆菌，已确定 NleB 具有糖基转移酶功能使用 N-乙酰氨基葡萄糖 (GlcNAc) 共价修饰宿主蛋白以破坏其正常状态的酶 NleB 破坏肿瘤坏死因子受体 (TNFR) 相关因子 2 (TRAF2) 信号传导，导致抑制促炎性 NF-κB 通路糖酵解酶 GAPDH 发挥辅助作用。 NleB 用 GlcNAc 修饰 GAPDH 可防止 GAPDH 与 TRAF2 活性结合。并激活 TRAF2，导致 NF-κB 对感染的反应降低，从而提高细菌的存活率。还发现一些 NleB 直向同源物修饰宿主蛋白的精氨酸残基，而不是修饰宿主蛋白的精氨酸残基。其丝氨酸/苏氨酸残基的预期修饰。鼠伤寒沙门氏菌菌株也编码 1-3 NleB。名为 SseK/1/2/3 的直向同源物，尽管它们的酶活性和对毒力的相对贡献较小清除。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，具体目标是：1）阐明NleB/SseK GlcNAcNAc糖基化机制。及其对宿主蛋白的靶向作用 2) 识别 GAPDH GlcNAc 酰化位点并表征其在其中的作用。 GAPDH 活性和 NF-κB 通路激活提供了一个机会。不仅要探讨这些毒力因子的酶学，还要影响小病毒的未来发展。具有抗毒特性的分子。