Regulating Microbial Biofilm Formation: A Novel Prokaryotic Multi-protein Complex

描述（由申请人提供）：本研究的长期目标是确定和了解铜绿假单胞菌利用的导致生物膜形成和受损个体慢性感染的精细调控机制的细节。铜绿假单胞菌是典型的机会致病菌。肺部的慢性铜绿假单胞菌感染是囊性纤维化患者死亡的最常见原因，强调了铜绿假单胞菌作为病原体的恶名。这种细菌能够在囊性纤维化肺等环境中定植和生存，并阻碍抗生素治疗和规避身体的免疫反应，这有许多关键。这些包括形成生物膜的能力，产生外粘多糖藻酸盐-天然屏障和毒力因子，作为一个社区通过多种群体感应系统协调生物反应的能力，以及感知和适应高氧化应激，铁限制和可用碳源条件的能力。毫不奇怪，这些属性紧密交织在一起，共同反映了铜绿假单胞菌调控网络的复杂性和优雅性。我们已经发现了一种新的多蛋白质复合物在铜绿假单胞菌，包括蛋白质组成的群体感应，海藻酸盐生物合成调节，氧化应激反应和响应环境铁和碳，包括新的蛋白质AlgH的功能还不清楚。基于复合物中蛋白质的特性，并且基于这些应答对于囊性纤维化肺中的生物膜形成和定殖和存活是不可或缺的事实，其功能是整合对各种关键生物信号的应答，并且对于囊性纤维化肺中的存活是必不可少的。此外，由于这些系统（群体感应系统、藻酸盐生物合成调节系统等）在生物学中的重要性，是控制铜绿假单胞菌感染的良好靶标，靶向这种新型蛋白质复合物在药学上应该是非常有利的，因为多个系统将同时被破坏。我们的总体假设是，铜绿假单胞菌作为生物膜在囊性纤维化肺中生存和生长所使用的复杂的调节和适应机制可以通过调节这种多蛋白复合物的形成/功能来破坏和控制。为了开始解决这个广泛的假设，我们建议：1）建立多蛋白质复合物形成的要求，2）确定多蛋白质复合物中的蛋白质间相互作用，并确定复合物的结构拓扑，3）解决多蛋白质复合物的组分蛋白质的结构在高分辨率下，和4）启动复合物的功能研究。研究结果应该为预防和治疗形成生物膜的细菌感染提供重要的新策略。

DESCRIPTION (provided by applicant): The long term goals of the research are to identify and understand the details of the elaborate regulatory mechanisms utilized by Pseudomonas aeruginosa that result in biofilm formation and chronic infections of compromised individuals. Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Chronic Pseudomonas aeruginosa infections of the lung are the most common cause of death in cystic fibrosis patients, underscoring the notoriety of Pseudomonas aeruginosa as a pathogen. There are many keys to the ability of this bacterium to colonize and survive in environments such as the cystic fibrosis lung and to thwart antibiotic regimes and circumvent the body's immune responses. These include the ability to form biofilms, to produce the exomucopolysaccharide alginate - a natural barrier and virulence factor, the ability to act as a community to coordinate biological responses via multiple quorum sensing systems, and the ability to sense and adapt to conditions of high oxidative stress, iron limitation, and available carbon sources. Not surprisingly, these attributes are tightly intertwined, and together reflect the complexity and elegance of the regulatory network in Pseudomonas aeruginosa. We have discovered a novel multi-protein complex in Pseudomonas aeruginosa that includes proteins integral to quorum sensing, alginate biosynthesis regulation, the oxidative stress response and the response to environmental iron and carbon, including the novel protein AlgH whose function is not understood. Based on the identities of the proteins in the complex, and based on fact that these responses are integral to biofilm formation and colonization and survival in the cystic fibrosis lung, it functions to integrate the responses to a variety of key biological signals and is essential for survival in the cystic fibrosis lung. Furthermore, because these systems (the quorum sensing system(s), the alginate biosynthesis regulatory system, etc.) are good targets for controlling Pseudomonas aeruginosa infections, targeting this novel protein complex pharmaceutically should be quite advantageous as multiple systems will simultaneously be disrupted. Our overall hypothesis is that the intricate regulatory and adaptive mechanisms used by Pseudomonas aeruginosa to survive and thrive in the cystic fibrosis lung as biofilms can be disrupted and controlled by regulating the formation/functions) of this multi-protein complex. To begin to address this broad hypothesis, we propose to; 1) Establish the requirements for formation of the multi-protein complex, 2) Identify the interprotein interactions in the multi-protein complex and determine the structural topology of the complex, 3) Solve the structures of component proteins of the multi-protein complex at high resolution, and 4) Initiate functional studies of the complex. The results of the research should provide important new strategies for prevention and treatment of infections by bacteria that form biofilms.