Comparative structural and functional analysis of Pseudomonas aeruginosa inhibitor of vertebrate lysozyme paralogs

脊椎动物溶菌酶旁系同源物铜绿假单胞菌抑制剂的比较结构和功能分析

• 批准号: 10410804
• 负责人: THOMAS C LEEPER
• 金额: $ 13.55万
• 依托单位: KENNESAW 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: 2022-08-26 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10410804
• 关键词: Antibiotics; Bacteria; Binding; Biochemical Pathway; Biochemistry; Biological Assay; Biophysics; Cell Wall; Complement; Complement Inactivators; Complex; Crosslinker; Cystic Fibrosis; Data Collection; Development; Drug Design; ESKAPE pathogens; Enzymatic Biochemistry; Enzyme Inhibition; Enzyme Kinetics; Enzymes; Family; Future; Genes; Glycoside Hydrolases; Human; Hydrolase; Hydrolysis; Immunity; In Vitro; Interferometry; Knock-out; Lytic; Mediating; Methods; Microbial Genetics; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Muramidase; Opportunistic Infections; Organism; Pathogenicity; Peptidoglycan; Phenotype; Plasmids; Property; Protein Isoforms; Proteins; Pseudomonas aeruginosa; Research; Resistance; Role; Small Interfering RNA; Specificity; Structure; Training; Translational Repression; Underrepresented Populations; Universities; Vertebral column; Work; antimicrobial; antimicrobial drug; comparative; crosslink; cystic fibrosis patients; drug discovery; experimental study; fighting; graduate student; in vivo; inhibitor; innovation; interest; knock-down; liquid chromatography mass spectrometry; member; molecular recognition; mortality; mutant; novel; novel drug class; opportunistic pathogen; paralogous gene; pathogen; pathogenic bacteria; repaired; structural biology; undergraduate student

Project Summary – Comparative structural and functional analysis of Pseudomonas aeruginosa inhibitor of vertebrate lysozyme paralogs. Thomas Leeper, PI, Kennesaw State University. The proposed integrative studies will investigate a significant regulator of peptidoglycan biochemistry in the cystic fibrosis pathogen Pseudomonas aeruginosa (PA), known to display multidrug resistance (MDR). Specifically, Inhibitor of Vertebrate Lysozyme protein 2 (IVYp2) will have its structure determined by NMR (Aim 1) and compared to its ability to inhibit hydrolase enzyme kinetics (Aim 2). While IVY proteins were originally suggested to inhibit lysozyme glycoside hydrolases, subsequent work suggested that the evolutionary origin of IVY’s may have been to inhibit lytic transglycosylases (LTs). For example, IVYp2 has no effect on lysozyme activity in enzyme inhibition assays but has been shown to inhibit LTs, at least in vitro. In order to determine which LTs are recognized in vivo enzyme inhibition assays with LTs (Aim 2) will be compared to novel applications of complementation and genetically encoded crosslinking studies in IVY knockout PA strains (Aim 3). The identified cognate IVY-LT pairs will be compared to the NMR structure of IVYp2 (Aim 1) and existing complex structures for other IVYs (i.e. IVYp1) bound to lysozyme to suggest regions important for LT molecular recognition. Graduate and undergraduate students, particularly those from underrepresented groups, will significantly participate in this research to facilitate data collection while providing them important training and advancement opportunities. These innovative studies will establish the background and workflow needed for future drug discovery efforts to develop new drug classes targeting novel biochemical pathways in MDR PA.

项目总结-铜绿假单胞菌抑制剂的比较结构和功能分析 脊椎动物溶菌酶的旁系同源物。 托马斯利珀，PI，肯尼索州立大学。 拟议的综合性研究将调查一个重要的调节肽聚糖的生物化学， 囊性纤维化病原体铜绿假单胞菌（PA），已知显示多药耐药 （医疗器械报告）。具体地，脊椎动物溶菌酶蛋白2（IVYp 2）的抑制剂将确定其结构 通过NMR（目的1）测定，并与其抑制水解酶动力学的能力（目的2）进行比较。虽然IVY 蛋白质最初被认为是抑制溶菌酶糖苷水解酶，随后的工作表明， IVY的进化起源可能是抑制溶解性转糖基酶（LT）。比如说， IVYp 2在酶抑制测定中对溶菌酶活性没有影响，但已显示抑制LT， 至少在试管中是这样。为了确定哪些LT在体内酶抑制试验中被识别， LT（目标2）将与互补和遗传编码的新应用进行比较。 IVY敲除PA菌株中的交联研究（Aim 3）。确定的同源IVY-LT对将 与IVYp 2的NMR结构（Aim 1）和其他IVY的现有复杂结构（即， IVYp 1）结合到溶菌酶，提示LT分子识别的重要区域。研究生和 本科生，特别是那些来自代表性不足的群体，将大大参与 这项研究，以促进数据收集，同时为他们提供重要的培训和进步 机会这些创新的研究将建立背景和工作流程所需的未来 药物发现努力开发靶向MDR PA中新生化途径的新药类别。