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Expanding the genetic code in Salmonella

扩展沙门氏菌的遗传密码

基本信息

批准号：
9334539
负责人：
Chenguang Fan
金额：
$ 13.65万
依托单位：
UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
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：
2016-08-18 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9334539
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 Health 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 fluorescence imaging functional group microbial novel pathogen pathogenic bacteria practical application protein activation protein complex segregation unnatural amino acids

项目摘要

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（由申请方提供）：沙门氏菌感染是一种常见的影响肠道的细菌性疾病。据估计，每年在美国造成约120万人患病，约23，000人住院治疗，450人死亡。由于多重耐药沙门氏菌菌株的出现，对其生理学和发病机制的深入了解对于确定新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将各种非天然氨基酸（UAA）整合到靶蛋白中，以解决由于20种天然氨基酸的化学多样性有限而难以或不可能通过大多数经典方法解决的问题。然而，很少有研究报告将UAA纳入人类病原体，如沙门氏菌。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掺入靶蛋白中，以形成用于共聚焦显微镜、顺磁共振光谱（EPR）、傅立叶变换红外光谱（FTIR）或荧光共振能量转移（FRET）的标记和探针（Aim 1）。为了验证概念，我们将使用这个OTS来研究沙门氏菌中细菌微区室（BMC）的空间组织，这是以前没有研究过的（目标2）。BMC是含有病毒衣壳样外壳和参与各种代谢途径的包封酶的大型蛋白质复合物。研究表明，1，2-丙二醇和乙醇胺降解，发生在BMC内，沙门氏菌的发病机制。我们将对叠氮基苯丙氨酸掺入沙门氏菌BMC的外壳蛋白中，然后通过无Cu点击反应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。将确定BMC的空间分布、细胞分裂期间BMC的分离、负责空间组织的BMC的关键蛋白以及BMC蛋白与细胞骨架组分之间的相互作用。这些研究将提供新的信息，并可能揭示新的范式，在我们的知识的BMC。总之，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式病原体，我们在这个建议中开发的OTS可以被许多实验室广泛使用，以促进微生物致病性的研究。