Mechanism and regulation of c-di-GMP signaling in bacterial biofilm formation

描述(申请人提供)：据估计，高达80%的慢性感染可归因于生物膜的形成，在生物膜形成过程中，细菌细胞变得固着，嵌入细胞外基质，最终形成一个大菌落。生物膜群落中的细胞往往逃脱了传统抗生素的治疗，并加剧了疾病的进程。生物膜的形成是一个复杂的、高度调控的过程，涉及到一个中央第二信使--环二GMP(c-di-GMP)，它控制着分化过程中的许多关键事件。潜在的信号机制和途径在很大程度上是未知的。环二GMP及其产生和降解的酶是真细菌所特有的，因此是开发抗细菌感染的新疗法的有吸引力的目标，这是拟议工作的长期目标。我们将着手研究生物膜形成过程中关键酶的结构、功能和调控。在特定的目标1和2中，我们分别关注催化c-di-GMP合成和周转的环化酶和磷酸二酯酶的不同亚家族。X射线结晶学和小角散射结合酶分析将被用来破译这些多结构域蛋白质的激活和调节机制。对构象可塑性和调控的深入了解对于小分子抑制剂的设计和优化将是非常有价值的。具体目标3侧重于通过使用蛋白质组学和遗传筛选来识别蛋白质-蛋白质相互作用来揭示信号通路和调节因子。这些研究的结果将阐明控制生物膜形成的基本信号反应和网络，并将为双化学和面向途径的小分子筛选提供基础。 传染病是发达国家死亡的主要原因之一。持续的慢性感染与细菌停留在自然表面(如心脏瓣膜、肺或耳朵)或医疗设备(如导管、植入物)并嵌入所谓的生物膜有关，给传统的抗菌治疗造成了不可逾越的障碍。这个过程似乎是高度受控的，通过研究酶和潜在的调节原理，我们希望为治疗学的发展提供新的起点。

DESCRIPTION (provided by applicant): It has been estimated that up to 80% of chronic infections can be attributed to biofilm formation, a differentiation process in which bacterial cells become sessile, embed themselves in an extracellular matrix and finally form a macrocolony. Cells within biofilm communities often escape treatment with traditional antibiotics and aggravate the course of disease. Biofilm formation is a complex and highly regulated process involving a central second messenger, cyclic di-GMP (c-di-GMP), that controls many of the key events during differentiation. The underlying signaling mechanisms and pathways are largely unknown. Cyclic di-GMP and enzymes for its production and degradation are unique to eubacteria, and therefore represent attractive targets for the development of novel therapeutics against bacterial infections, a long-term goal of the proposed work. We will set out to study the structure, function and regulation of key enzymes in biofilm formation. In Specific Aims 1 and 2, we focus on distinct subfamilies of cyclases and phosphodiesterases that catalyze the synthesis and turnover of c-di-GMP, respectively. X-ray crystallography and small-angle scattering in combination with enzymatic assays will be used to decipher the activation and regulatory mechanisms of these multi-domain proteins. Insight into the conformational plasticity and regulation will be invaluable for the design and optimization of small molecule inhibitors. Specific Aim 3 focuses on uncovering signaling pathways and regulators by the identification of protein-protein interactions using proteomics and genetic screens. The results of these studies will elucidate the basic signaling reactions and networks that control biofilm formation, and will provide the basis for bicochemical and pathway-oriented small molecule screens. Infectious diseases are one of the foremost causes of mortality in developed countries. Persistent chronic infections have been associated with a phenomenon in which bacteria settle down on natural surfaces (e.g. heart valves, lungs, or ears) or medical devices (e.g. catheters, implants) and embed themselves in a so called biofilm, causing insuperable obstacles for traditional antimicrobial treatments. The process appears to be highly controlled, and by studying the enzymes and underlying regulatory principles we hope to provide novel starting points for the development of therapeutics.