Expanding the genetic code in Salmonella

扩展沙门氏菌的遗传密码

• 批准号: 8950306
• 负责人: Chenguang Fan
• 金额: $ 24.98万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8950306
• 关键词: Address; Affect; Affinity; Amino Acids; Applied Genetics; Bacteria; Bacterial Infections; Bacterial Proteins; Biological Assay; Capsid; Cell Cycle; Cell division; Cells; Cessation of life; Chemicals; Chemistry; Confocal Microscopy; Cytoskeletal Proteins; Electron Spin Resonance Spectroscopy; Encapsulated; Enzymes; Escherichia coli; Ethanolamines; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Genes; Genetic Code; Green Fluorescent Proteins; Hospitalization; Human; Image; Intestines; Knock-out; Knowledge; Label; Laboratories; Life; Link; Maps; Mass Spectrum Analysis; Metabolic Pathway; Methods; Modeling; Monitor; Multi-Drug Resistance; Organism; Pathogenesis; Phenylalanine; Physiology; Positioning Attribute; Post-Translational Protein Processing; Problem Solving; Propylene Glycols; Proteins; Reaction; Reporting; Research; Salmonella; Salmonella infections; Scientist; Spatial Distribution; Spectroscopy, Fourier Transform Infrared; Structure; Substrate Specificity; System; Testing; Translations; Tyrosine-tRNA Ligase; United States; Variant; Viral; antimicrobial drug; base; functional group; microbial; novel; pathogen; pathogenic bacteria; practical application; protein activation; protein complex; public health relevance; segregation

 DESCRIPTION (provided by applicant): Salmonella infection is a common bacterial disease that affects the intestinal tract. It is estimated to cause about 1.2 million illnesses in the Unitd States each year, with about 23,000 hospitalizations and 450 deaths. A deep knowledge of its physiology and pathogenesis is important for identifying novel targets to develop new antimicrobial agents due to the emergence of multidrug-resistant Salmonella strains. Currently, the genetic code expansion strategy has been widely used to incorporate various unnatural amino acids (UAAs) into target proteins to solve the problems which are difficult or impossible to address by most classical methods due to the limited chemical diversity of the 20 natural amino acids. However, very few studies have been reported to incorporate UAAs in human pathogens such as Salmonella. Here, we will expand the genetic code in Salmonella by introducing an orthogonal translation system (OTS) which can efficiently incorporate various UAAs into target proteins to form labels and probes for confocal microscopy, paramagnetic resonance spectroscopy (EPR), Fourier transform infrared spectroscopy (FTIR), or fluorescence resonance energy transfer (FRET) (Aim 1). For proof-of-concept, we will use this OTS to investigate the spatial organization of bacterial microcompartments (BMCs) in Salmonella which has not been studied before (Aim 2). BMCs are large protein complexes containing viral capsid-like shells and encapsulated enzymes involved in various metabolic pathways. Studies have implicated 1,2-propanediol and ethanolamine degradations, which occur within BMCs, in Salmonella pathogenesis. We will incorporate p-azido-phenylalanine into the shell proteins of BMCs in Salmonella, followed by a Cu-free click reaction to form small-sized fluorescent probes for dynamic imaging to overcome the potential interference with the assemblies and functions of BMCs from the commonly used fluorescent protein tags. The spatial distribution of BMCs, the segregation of BMCs during cell divisions, key proteins of BMCs responsible for the spatial organization, and the interactions between BMC proteins and cytoskeletal components will be determined. These studies will provide new information and may reveal new paradigms in our knowledge of BMCs. In summary, we will show a practical application of the genetic code expansion strategy in studies of bacteria. Since Salmonella is an important model pathogen, the OTS we developed in this proposal could be broadly used by many laboratories to facilitate studies of microbial pathogenesis.

 描述（由适用提供）：沙门氏菌感染是一种影响肠道的常见细菌疾病。据估计，每年的单位州造成约120万疾病，约有23,000例住院和450例死亡。由于多药耐药的沙门氏菌菌株的出现，人们对其生理和发病机理的深入了解对于确定开发新抗菌剂的新靶标很重要。当前，遗传密码扩展策略已被广泛用于将各种非天然氨基酸（UAAS）纳入靶蛋白中，以解决由于20种天然氨基酸的化学多样性有限，因此大多数经典方法难以解决的问题。但是，据报道，很少有研究将UAA纳入人类病原体，例如沙门氏菌。在这里，我们将通过引入正交翻译系统（OT）来扩展沙门氏菌中的遗传密码，该系统可以有效地将各种UAA纳入靶蛋白中，以形成共聚焦显微镜，参数磁共振光谱光谱镜检查（EPR）的标签和问题，傅立叶感染光谱（FTIR）或荧光能量能量转移（FTIR）或荧光转移（frece Resenance aim）（fret）（FIRE）（FIRE）（FIRE）（FIRE）（FIRET）（FIRET）（FIRET）（FIRET）（FRET）（FRET）。为了证明概念概念，我们将使用这种OT来调查沙门氏菌中细菌微校区（BMC）的空间组织，我们还没有将P-氮杂 - 苯丙氨酸掺入BMC的壳蛋白中，以鲑鱼的壳蛋白中的壳蛋白，以无效的单击反应，以使小型荧光概念构成动态概率的反应。来自常用的荧光蛋白标签。将确定BMC的空间分布，细胞分裂过程中BMC的分离，负责空间组织的BMC的关键蛋白质以及BMCS蛋白与细胞骨架成分之间的相互作用。这些研究将提供新的信息，并可能揭示我们对BMC的知识中的新范例。总而言之，我们将在细菌研究中展示遗传代码扩展策略的实际应用。由于沙门氏菌是一种重要的模型病原体，因此我们在该提案中开发的OT可以广泛使用许多实验室来促进微生物发病机理的研究。