Mechanistic studies on peptidoglycan tailoring that mediate biofilm stability

描述（由申请人提供）：防止生物膜聚集或促进生物膜扩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）负责将非规范d -氨基酸结合到枯草芽孢杆菌的肽聚糖中，并且一旦结合，在生物膜稳定性中发挥作用。通过这项工作，我们希望通过酶降解、色谱、质谱分析和与化学合成标准的比较，在体内证明非规范d -氨基酸，特别是D-Tyr，被纳入枯草芽孢杆菌肽聚糖的五肽中。我们还将利用化学合成的脂质II底物和从枯草芽孢杆菌中克隆的PBP1a和1b来评估它们的掺入效率，从而证明枯草芽孢杆菌pp1a和1b可以在体外发挥这一功能。单个基因的突变影响

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.