T3SS Effector Regulation of Bacterial Metabolism

T3SS 细菌代谢的效应器调节

• 批准号: 10612897
• 负责人: Derek Mosier
• 金额: $ 22.43万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-22 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10612897
• 关键词: Affect; Area; Arginine; Bacterial Physiology; Bacterial Proteins; Behavior; Biochemical; Biological Assay; Cells; Citrobacter rodentium; Cyclic AMP Receptor Protein; DNA; DNA Binding; DNA Damage; Data; Electrophoretic Mobility Shift Assay; Enterocytes; Enzymes; Escherichia coli; Escherichia coli EHEC; Family; Funding Mechanisms; Future; Gene Expression; Gene Expression Regulation; Genes; Glucosamine; Glutathione; Goals; Gram-Negative Bacteria; Inflammatory; Innate Immune System; Investigation; Mammalian Cell; Manuscripts; Mass Spectrum Analysis; Measurement; Mediating; Metabolism; Molecular Conformation; NF-kappa B; Names; Orthologous Gene; Oxidative Stress; Pathogenicity Island; Pathway interactions; Play; Post-Translational Protein Processing; Production; Protein Glycosylation; Proteins; Publishing; Pyruvaldehyde; Regulation; Regulon; Reporter; Repressor Proteins; Research; Role; Salmonella; Salmonella enterica; Stress; Structure; Surface Plasmon Resonance; System; Testing; Type III Secretion System Pathway; Virulence; Work; bacterial metabolism; complex biological systems; experimental study; glutathione synthase; glycation; glycosylation; glycosyltransferase; improved; innovation; insight; novel; pathogenic bacteria; transcription factor; transcriptome; transcriptome sequencing; uptake; virulence gene

Project Summary. Many Gram-negative bacterial pathogens interact with mammalian cells by using secretion systems to inject virulence proteins directly into infected host cells. Some of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of mammalian proteins by catalyzing the addition of unusual post- translational modifications. Type III secretion system (T3SS) effectors play essential roles in virulence and their mechanisms have provided great insight into the functions and components of the innate immune system. T3SS effectors are believed to be inactive until they are injected into host cells, where they then fold into their active conformations. However, recent work with the NleB and SseK glycosyltransferases from E. coli, Citrobacter rodentium, and Salmonella enterica has challenged that dogma. NleB glycosylates and activates the bacterial glutathione synthetase (GshB) enzyme, resulting in enhanced glutathione production and improved C. rodentium survival in oxidative stress conditions. SseK1 is active within Salmonella enterica, where it glycosylates and enhances the activity of several enzymes that are critical to the ability of Salmonella to resist methylglyoxal stress. In support of long-term goals to identify and understand the functional significance of bacterial protein glycosylation by NleB and SseK1, two new bacterial targets of SseK1, namely NagC and CRP have been discovered. NagC is a dual activator-repressor that controls GlcNAc uptake and metabolism. NagC also regulates locus of enterocyte effacement (LEE) gene expression in enterohemorrhagic E. coli (EHEC). The LEE is an important pathogenicity island that encodes the T3SS and many effector proteins. The catabolite repressor protein [(CRP); also referred to as the catabolite activator protein (CAP)], is a global regulator that mediates the expression of ~150 genes, including those important to GlcNAc metabolism and several T3SS components in Salmonella. The central hypothesis to be tested is that Arg-glycosylation of NagC and CRP by SseK1 affects Salmonella virulence gene regulation and metabolism. The specific aims are: 1) Quantify the extent to which Arg-glycosylation of NagC and CRP affects the ability of these transcription factors to bind DNA; 2) Characterize the impact of Arg-glycosylation of NagC and CRP on the Salmonella transcriptome. The proposed work is highly suitable for the R21 funding mechanism because of the innovation of the research premise and novel hypothesis to be tested. Such data will establish the framework for future investigation of the mechanistic aspects and functional significance of T3SS effector regulation of bacterial transcription factors.

项目摘要。 许多革兰氏阴性细菌病原体通过使用分泌系统进行注射与哺乳动物细胞相互作用 病毒蛋白直接成被感染的宿主细胞。这些注入的蛋白质“效应子”中的一些是酶 修改结构并抑制哺乳动物蛋白质的功能，通过催化不寻常的后加入 翻译修改。 III型分泌系统（T3SS）生效器在病毒及其中起着重要的作用 机制为先天免疫系统的功能和组成部分提供了深入的了解。 T3SS 效果被认为是不活跃的，直到将它们注入宿主细胞为止，然后它们折叠到活性 构象。但是，最近与大肠杆菌的NLEB和SSEK糖基转移酶进行的工作 Rodentium和Salmonella Enterica对教条提出了挑战。 NLEB糖基化并激活细菌 谷胱甘肽合成酶（GSHB）酶，导致谷胱甘肽产生增强并改善了啮齿动物念珠菌 在氧化应激条件下的生存。 ssek1在肠沙门氏菌中活跃，在那里它糖基化和 增强了几种酶的活性，这些酶对沙门氏菌抗甲基糖的能力至关重要 压力。支持长期目标以识别和理解细菌蛋白的功能意义 NLEB和SSEK1糖基化，SSEK1的两个新细菌靶标，即NAGC和CRP 发现。 NAGC是一种双重激活剂抑制器，可控制GlcNAC摄取和代谢。 NAGC也是如此 调节肠肠癌大肠杆菌（EHEC）中肠细胞能量（LEE）基因表达的轨迹。李 是编码T3SS和许多效应蛋白的重要致病岛。分解代谢物复制品 蛋白质[（CRP）;也称为分解代谢物激活剂蛋白（CAP）]，是一个介导的全球调节剂 〜150个基因的表达，包括对GlcNAC代谢重要的基因和在 沙门氏菌。要测试的中心假设是SSEK1对NAGC和CRP的ARG-糖基化影响 沙门氏菌病毒基因调节和代谢。具体目的是：1）量化 NAGC和CRP的ARG-糖基化会影响这些转录因子结合DNA的能力。 2）表征 NAGC和CRP的ARG-糖基化对沙门氏菌转录组的影响。拟议的工作很高 由于研究前提和新假设的创新，适用于R21资金机制 要测试。这样的数据将建立机械方面未来投资的框架和 T3SS效应子调节细菌转录因子的功能意义。