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（由申请人提供）：沙门氏菌感染是一种影响肠道的常见细菌性疾病，估计每年在美国导致约 120 万人患病，其中约 23,000 人住院，450 人死亡。对其生理学的深入了解。由于多重耐药沙门氏菌菌株的出现，发病机制对于确定新的靶点以开发新的抗菌药物非常重要。目前，遗传密码扩展策略已被广泛用于治疗。由于 20 种天然氨基酸的化学多样性有限，将各种非天然氨基酸 (UAA) 掺入目标蛋白中，以解决大多数经典方法难以或不可能解决的问题。然而，很少有研究报道掺入 UAA。在这里，我们将通过引入正交翻译系统（OTS）来扩展沙门氏菌的遗传密码，该系统可以有效地将各种 UAA 整合到目标蛋白中，形成用于共聚焦显微镜的标记和探针，顺磁共振光谱 (EPR)、傅里叶变换红外光谱 (FTIR) 或荧光共振能量转移 (FRET)（目标 1），我们将使用此 OTS 来研究细菌微区室 (BMC) 的空间组织。 ）在沙门氏菌中尚未被研究过（目标 2）。 BMC 是含有病毒衣壳样外壳和参与各种代谢的封装酶的大型蛋白质复合物。研究表明，BMC 内发生的 1,2-丙二醇和乙醇胺降解与沙门氏菌发病机制有关。我们将对叠氮基苯丙氨酸掺入沙门氏菌 BMC 的壳蛋白中，然后进行无铜点击反应以形成。用于动态成像的小尺寸荧光探针，以克服常用荧光蛋白标签对 BMC 组装和功能的潜在干扰。细胞分裂过程中 BMC 的分离、负责空间组织的 BMC 关键蛋白以及 BMC 蛋白和细胞骨架成分之间的相互作用将被确定，这些研究将提供新的信息，并可能揭示我们对 BMC 的认识的新范例。我们将展示遗传密码扩展策略在细菌研究中的实际应用，由于沙门氏菌是一种重要的模型病原体，因此我们在本提案中开发的 OTS 可以被许多实验室广泛使用，以促进微生物发病机制的研究。