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的形成，这是一个分化过程，细菌细胞变得无根，将自己嵌入细胞外基质中，最终形成一个大菌落。生物膜群落内的细胞常常逃避传统抗生素的治疗，从而加重疾病的进程。生物膜的形成是一个复杂且高度调控的过程，涉及中心第二信使环二gmp (c-di-GMP)，它控制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及其生产和降解酶是真细菌所特有的，因此代表了开发抗细菌感染的新疗法的有吸引力的靶点，这是提出的工作的长期目标。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Specific Aims 1和2中，我们重点研究了环化酶和磷酸二酯酶的不同亚家族，它们分别催化c-di-GMP的合成和转化。x射线晶体学和小角度散射结合酶分析将用于破译这些多结构域蛋白的激活和调节机制。深入了解小分子抑制剂的构象可塑性和调控将对小分子抑制剂的设计和优化具有不可估量的价值。特异性目标3侧重于通过蛋白质组学和遗传筛选鉴定蛋白质相互作用来揭示信号通路和调节因子。这些研究结果将阐明控制生物膜形成的基本信号反应和网络，并将为生化和途径导向的小分子筛选提供基础。

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.