The role of proteolysis in bacterial biofilm formation

 描述(申请人提供)：细菌生物膜，由表面附着的细菌产生的结构，是对抗生素耐药的感染的基础。霍乱弧菌是引起腹泻疾病霍乱的革兰氏阴性细菌，这些结构有助于附着在环境表面。理解这些结构形成的过程对于阻止这一过程是至关重要的。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对特定的环境条件做出反应。这种基质由VPS胞外多糖、蛋白质和DNA组成。通过对这一基质的蛋白质组学分析，我们最近鉴定了三种分泌蛋白，BAP1，RbmC和RBMA，它们是霍乱弧菌生物膜结构完整性所必需的。BAP1和RbmC集中在生物膜和基质之间，介导生物膜结构与表面的粘连。相反，RBMA分散在整个生物膜中，并包围着与生物膜相关的细胞。根据结构数据，RBMA被假设结合细菌O抗原和VPS多糖，将生物膜基质拉到细菌细胞表面。在我们的研究中，我们注意到RBMA在成熟的生物膜中经历了蛋白水解性切割。我们的初步结果表明，RBMA的过早切割增加了细胞对生物膜的招募。在这个应用中，我们建议评估一个模型，在该模型中，切割增加了RBMA对O抗原和VPS多糖的亲和力，从而一旦细胞在生物膜内生长完成，就“锁定”生物膜结构。我们将通过鉴定RBMA蛋白水解酶，阐明调节RBMA蛋白分解的机制，并探索RBMA蛋白分解对RBMA功能和成熟生物膜对机械应力的抗性的影响来检验这一模型。这些研究将为蛋白质分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这一过程中生物膜基质的强化。

 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.