The role of proteolysis in bacterial biofilm formation

蛋白水解在细菌生物膜形成中的作用

• 批准号: 8807275
• 负责人: PAULA I WATNICK
• 金额: $ 26.5万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8807275
• 关键词: Adherence; Adhesives; Affinity; Antibiotics; Antigens; Bacteria; Binding; Biochemical; Biological; Cell surface; Cells; Cholera; Cleaved cell; DNA; Data; Disease; Enzymes; Extracellular Matrix; Gram-Negative Bacteria; Infection; Length; Ligands; Mechanical Stress; Mediating; Microbial Biofilms; Modeling; Molecular; Mutagenesis; O Antigens; Peptide Hydrolases; Phase; Polysaccharides; Process; Proteins; Proteolysis; Proteomics; Psychological reinforcement; Resistance; Role; Structure; Surface; Techniques; Tetanus Helper Peptide; Vibrio cholerae; Whole Cell Vaccine; base; cell growth; improved; new technology; overexpression; premature; prevent; public health relevance

 DESCRIPTION (provided by applicant): Bacterial biofilms, the structures created by surface attached bacteria, are the basis of infections that are recalcitrant to antibiotics. For Vibrio cholerae, the Gram negative bacterium that causes the diarrheal disease cholera, these structures facilitate attachment to environmental surfaces. An understanding of the process by which these structures form is essential to blocking the process. V. cholerae responds to specific environmental conditions by synthesizing an adhesive extracellular matrix that promotes biofilm formation. This matrix is comprised of the VPS exopolysaccharide, proteins, and DNA. Through a proteomic analysis of this matrix, we recently identified three secreted proteins, Bap1, RbmC, and RbmA, that are required for the structural integrity of the V. cholerae biofilm. Bap1 and RbmC, which are concentrated between the biofilm and the substratum, mediate adherence of the biofilm structure to the surface. In contrast, RbmA is dispersed throughout the biofilm and surrounds biofilm-associated cells. Based on structural data, RbmA is hypothesized to bind both the bacterial O-antigen and VPS polysaccharide, pulling the biofilm matrix onto the bacterial cell surface. During our studies, we noted that RbmA undergoes proteolytic cleavage in mature biofilms. Our preliminary results suggest that premature cleavage of RbmA augments recruitment of cells to the biofilm. In this application, we propose to evaluate a model in which cleavage increases the affinity of RbmA both for the O-antigen and the VPS polysaccharides, thus "locking in" the biofilm structure once cell growth within the biofilm is complete. We will tet this model by identifying the RbmA protease or proteases, elucidating the mechanisms by which proteolysis of RbmA is regulated, and exploring the impact of RbmA proteolysis on RbmA function and resistance of mature biofilms to mechanical stress. These studies will define a new paradigm for the role of proteolysis in bacterial biofilm maturation and may suggest new technologies to prevent reinforcement of the biofilm matrix during this process.

 描述（由申请人提供）：细菌生物膜，即表面附着细菌产生的结构，是抗生素无法抵抗的感染的基础。对于霍乱弧菌，革兰氏阴性细菌，导致霍乱，这些结构有助于附着在环境表面。了解这些结构形成的过程对于阻止这一过程至关重要。霍乱弧菌通过合成促进生物膜形成的粘附性细胞外基质来响应特定的环境条件。该基质由VPS胞外多糖、蛋白质和DNA组成。通过对该基质的蛋白质组学分析，我们最近确定了三种分泌蛋白，Bap 1，RbmC和RbmA，它们是霍乱弧菌生物膜结构完整性所必需的。Bap 1和RbmC，集中在生物膜和基质之间，介导生物膜结构粘附到表面。相反，RbmA分散在整个生物膜中并包围生物膜相关细胞。基于结构数据，假设RbmA结合细菌0-抗原和VPS多糖两者，将生物膜基质拉到细菌细胞表面上。在我们的研究期间，我们注意到RbmA在成熟生物膜中经历蛋白水解裂解。我们的初步结果表明，过早切割的RbmA增强招聘的细胞的生物膜。在本申请中，我们建议评估一种模型，其中切割增加了RbmA对O-抗原和VPS多糖的亲和力，从而一旦生物膜内的细胞生长完成，就“锁定”生物膜结构。我们将通过鉴定RbmA蛋白酶或蛋白酶，阐明RbmA蛋白水解的调节机制，并探索RbmA蛋白水解对RbmA功能和成熟生物膜对机械应力的抵抗力的影响来泰特该模型。这些研究将为蛋白水解在细菌生物膜成熟中的作用定义一个新的范例，并可能提出新的技术来防止在此过程中生物膜基质的强化。