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扩散将加快生物膜相关感染的治疗。我们推测，青霉素结合蛋白(PBPs)负责将非规范的D-氨基酸掺入枯草芽孢杆菌的肽聚糖中，一旦掺入，就会对生物膜的稳定性起作用。通过这项拟议的工作，我们希望通过酶降解、层析、质谱学以及与化学合成标准的比较，在体内证明非规范的D-氨基酸，特别是D-Tyr被结合到枯草杆菌肽多聚糖的五肽中。我们还将证明枯草杆菌PBP1a和1b可以通过利用化学合成的脂质II底物和从枯草杆菌克隆的PBP1a和1b在体外发挥这一功能，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。我们预计最近在波纳发现的突变， 编码PBP 1a和1b的基因将产生突变的PBPs，这种突变的PBPs在将D-Tyr结合到肽聚糖中时效率低下。我们将克隆、高效表达和纯化突变的枯草杆菌PBPs，并测试掺入效率，以进一步验证我们的假设，即细胞壁剪裁负责生物膜调节。该工作为阐明D-氨基酸引发生物膜解离的机制奠定了基础。展示多溴联苯对生物膜的调节将允许开发治疗生物膜相关感染的新策略。 与公共卫生相关：传染病细菌形成生物膜，严重威胁公共健康。本研究旨在了解生物膜扩散的机理。更好地了解这些生物膜是如何解离的，可能会导致更好地对抗细菌感染的治疗策略。

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. PUBLIC HEALTH RELEVANCE: Infectious bacteria form biofilms that are a serious threat to public health. The research proposed here is directed toward understanding the mechanism of biofilm dispersal. A better understanding of how these biofilms dissociate might lead to therapeutic strategies to better combat bacterial infections.