新烯酰辅酶A水合酶/异构酶家族在致病铜绿假单胞菌群体感应信号分子合成中的分子机制

Pathogenic Pseudomonas aeruginosa is the major cause of persistent infection in lungs of cystic fibrosis (CF) patients, which lead to acute exacerbations of the lung disease. Quorum-sensing (QS) system is the primary regulation mechanism for virulence factor expression and secretion in Pseudomonas aeruginosa, synthesizing specific signal molecules and controlling bacteria flora behavior in response to the concentration changes of those signal molecules under microenvironments. Recently, it has been demonstrated that Pseudomonas aeruginosa produces a new type of QS signal molecule--cis-2-decenoic acid, this molecule is responsible for induction of the native biofilm dispersion and drug resistance, and its related pathway is considered as the new targets for anti-infection treatments and drug development, however, little is known about its synthesis and functional mechanisms. This work will focus on the enoyl-coenzyme A hydratase/isomerase family which is critical for unsaturated fatty acid secondary metabolisms in Pseudomonas aeruginosa. We have cloned and expressed 9 genes and proteins from this family, we are going to verify the cis-2-decenoic acid synthesis pathway by gene knock out and host cell infection model test, also we want to investigate their catalytic mechanisms and substrate recognition specificity based on protein structure analysis and biochemistry experiments, to provide basic theory and feasibility study for screening inhibitor against enoyl-coenzyme A hydratase/isomerase.

致病性铜绿假单胞菌对肺囊肿性纤维化患者的持续性感染是导致其肺组织病变加剧的主因.群体感应系统是铜绿假单胞菌致病因子表达与分泌的主要调控机制, 通过合成放特定的信号分子,并感知其在微环境中的浓度变化调节细菌的群体行为。 顺式二癸烯酸是近年来在铜绿假单胞菌中发现的新型群体感应信号分子，对调节其菌膜的扩散和耐药性非常重要，其相关信号通路被认为是抗感染治疗与药物研发的新靶点，但对其合成与作用机制还所知甚少。本课题以铜绿假单胞菌不饱和脂肪酸类分子次生代谢途径中的关键蛋白烯酰辅酶A水合酶/异构酶家族为研究对象, 我们克隆并表达了其9个成员的基因和蛋白，利用基因敲除和宿主细胞侵染模型，确定参与顺式二癸烯合成的通路，基于蛋白结构和生化实验探明催化机制和底物识别特异性，以期为基于烯酰辅酶A水合酶/异构酶为靶点的小分子抑制剂筛选提供理论基础和可行性分析。

铜绿假单胞菌是肺囊肿性纤维化患者最普遍感染又极难根除的病原体。铜绿假单胞菌表面的菌膜赋予其很强的环境适应性和耐药性，而不饱和脂肪酸类分子是菌膜形成过程中的关键信号分子。本课题的研究对象是铜绿假单胞菌不饱和脂肪酸类分子次生代谢途径中的关键酶烯酰辅酶A水合酶/异构酶家族蛋白的结构及功能。在本课题中，通过分子克隆，表达纯化，结晶筛选，并且基于X-射线衍射，得到了DspI(PA0745), PaECI(PA4980), OdaA(PA4330)三个蛋白的晶体结构。分析结构，构建了可能对其功能具有重要影响的突变体，并通过构建基因突变株，生化实验及DSF实验等证实了这些突变体的作用，探明其底物识别特异性。我们的研究对基于烯酰辅酶A水合酶/异构酶为靶点的小分子抑制剂筛选提供了一定的理论基础。