Mechanistic studies on peptidoglycan tailoring that mediate biofilm stability

描述（由申请人提供）：防止生物膜组装或促进生物膜分散将加快生物膜相关感染的治疗。我们假设，青霉素结合蛋白（PBPs）负责非典型的D-氨基酸掺入到枯草芽孢杆菌的肽聚糖，一旦纳入，发挥生物膜稳定性的作用。通过这项工作，我们希望能证明非典型的D-氨基酸，特别是D-酪氨酸，被纳入五肽的B。使用酶降解、色谱法、质谱法的组合并与化学合成的标准品比较，在体内测定枯草杆菌肽聚糖。我们还将证明B。枯草杆菌PBP 1a和1B可以利用化学合成的脂质II底物和从B克隆的PBP 1a和1B在体外执行该功能。subtilis以评估它们的掺入效率。单个基因的突变会影响 枯草芽孢杆菌生物膜的分解。我们希望最近发现的ponA突变， 编码PBP 1a和1b的基因将产生不能有效地将D-Tyr掺入肽聚糖的突变型PBP。我们将克隆、过表达和纯化突变体B。枯草芽孢杆菌PBP和测试掺入效率，以进一步验证我们的假设，即细胞壁剪裁是生物膜调节的原因。本研究为进一步阐明D-氨基酸触发生物膜解离的机制奠定了基础.通过PBPs证明生物膜调节将允许开发治疗生物膜相关感染的新策略。

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.