Mechanistic studies on peptidoglycan tailoring that mediate biofilm stability

介导生物膜稳定性的肽聚糖剪裁机制研究

• 批准号: 8514399
• 负责人: Matthew Dennis Lebar
• 金额: $ 5.22万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514399
• 关键词: Affect; Amino Acids; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Infections; Biochemical; Biological Assay; Catalytic Domain; Catheters; Cell Wall; Cells; Chromatography; Chronic; Complex; Cytolysis; Cytoplasm; Development; Disaccharides; Enzymes; Extracellular Matrix; Foundations; Genes; Health; Human; Implant; In Vitro; Infection; Lead; Link; Lipids; Mass Spectrum Analysis; Mediating; Medical Device; Microbial Biofilms; Monitor; Mutation; Osmotic Pressure; Penicillin-Binding Proteins; Peptides; Peptidoglycan; Peptidoglycan glycosyltransferase; Peptidyltransferase; Play; Polymers; Positioning Attribute; Process; Public Health; Radioactive; Regulation; Research; Resistance; Role; Route; Testing; Therapeutic; Work; analytical method; base; combat; crosslink; in vitro Assay; in vitro activity; in vivo; mutant; overexpression; polymerization; prevent; tool

DESCRIPTION (provided by applicant): Preventing biofilm assembly or facilitating biofilm dispersal would expedite treatment of biofilm-related infections. We hypothesize that penicillin-binding proteins (PBPs) are responsible for non-canonical D-amino acid incorporation into the peptidoglycan of Bacillus subtilis and, once incorporated, play a role in biofilm stability. Throug this proposed work, we expect to show that non-canonical D-amino acids, specifically D-Tyr, are incorporated into the pentapeptide of B. subtilis peptidoglycan in vivo using a combination of enzymatic degradation, chromatography, mass spectrometry, and comparison to chemically synthesized standards. We will also show that B. subtilis PBP 1a and 1b can perform this function in vitro by utilizing chemically synthesized lipid II substrate and cloned PBP1a and 1b from B. subtilis to assess their incorporation efficiency. Mutations in a single gene influence the disassembly of biofilms of Bacillus subtilis. We expect that recently identified mutations in ponA, the gene encoding for PBP 1a and 1b will give rise to mutant PBPs that are inefficient in incorporating D-Tyr into peptidoglycan. We will clone, over express, and purify the mutant B. subtilis PBPs and test incorporation efficiency to further validate our hypothesis that cell wall tailoring is responsible for biofilm regulation. This work will provide the foundation for elucidatng the mechanism of D- amino acid triggered biofilm disassociation. Demonstrating biofilm regulation by PBPs will allow for the development of new strategies toward treatment of biofilm related infections.

描述（由申请人提供）：防止生物膜组装或促进生物膜扩散将加快与生物膜相关感染的治疗。我们假设青霉素结合蛋白（PBP）负责将非典型的D-氨基酸掺入枯草芽孢杆菌的肽聚糖中，并且一旦纳入，就在生物膜稳定性中起作用。通过这项拟议的工作，我们期望表明非经典的D-氨基酸，特别是D-TYR，将枯草芽孢杆菌肽聚糖的五肽纳入体内五肽中，并使用酶促降解的组合，色谱，质谱和质谱和比较化学合成的标准。我们还将表明，枯草芽孢杆菌PBP 1A和1B可以通过使用化学合成的脂质II底物和克隆的PBP1A和枯草芽孢杆菌的1B来在体外执行此功能，以评估其掺入效率。单个基因中的突变影响 枯草芽孢杆菌生物膜的拆卸。我们希望最近发现Pona的突变 编码PBP 1A和1B的基因将产生突变的PBP，这些突变体PBP效率低下，将D-TYR掺入肽聚糖中。我们将克隆，过度表达并净化突变的枯草芽孢杆菌PBP，并进行测试掺入效率，以进一步验证我们的假设，即细胞壁裁缝负责生物膜调节。这项工作将为阐明D-氨基酸的机理触发生物膜分离的机理提供基础。证明PBP的生物膜调节将允许发展与生物膜相关感染的新策略。