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

描述（由申请人提供）：据估计，高达80%的慢性感染可归因于生物膜形成，生物膜形成是一种细菌细胞变得固着、将自身嵌入细胞外基质并最终形成大菌落的分化过程。生物膜群落中的细胞通常逃避传统抗生素的治疗，并加重疾病的进程。生物膜的形成是一个复杂的和高度调节的过程，涉及中央第二信使，环二GMP（c-di-GMP），控制分化过程中的许多关键事件。潜在的信号传导机制和途径在很大程度上是未知的。环状二GMP及其产生和降解的酶是真细菌所特有的，因此代表了开发抗细菌感染的新型治疗剂的有吸引力的靶点，这是所提出工作的长期目标。我们将着手研究生物膜形成中关键酶的结构、功能和调控。在具体目标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射线晶体学和小角散射结合酶测定将用于破译这些多结构域蛋白质的激活和调节机制。深入了解构象的可塑性和调控将是非常宝贵的小分子抑制剂的设计和优化。具体目标3侧重于通过使用蛋白质组学和遗传筛选鉴定蛋白质-蛋白质相互作用来揭示信号通路和调节剂。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，并将为双化学和路径导向的小分子筛选提供基础。 在发达国家，传染病是造成死亡的主要原因之一。持续性慢性感染与细菌定居在自然表面（例如心脏瓣膜、肺或耳朵）或医疗装置（例如导管、植入物）上并将其自身嵌入所谓的生物膜中的现象相关，这对传统的抗微生物治疗造成了无法克服的障碍。这一过程似乎是高度受控的，通过研究酶和潜在的调控原则，我们希望为治疗药物的开发提供新的起点。

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.