猪链球菌LuxS蛋白80,87位氨基酸突变对其结构及调控功能的影响

LuxS-based quorum sensing which produces cell signaling molecules autoinducer (AI)-2, has been proved to play an important role for biofilm formation, virulence and so on. In our previous project, encoding protein expressed in E.coli bacteria was obtained in vitro by using recombinant gene technology. Crystals suitable for X-ray diffraction was obtained by screening and optimizing, as well as the fine three dimensional structures were solved. S-Ribosylhomocysteinase (LuxS protein) from Streptococcus suis was initially identified by bioinformatics methods. Compared with LuxS protein amino acid sequence and three-dimensional structure of the other bacterium, we found that the LuxS protein have mutation near substrate binding 80 and 87 amino acid, suggesting that the site of mutations may be relevant to evolution mechanism of LuxS and regulate bacteria other physiological function. In the current project, the enzyme activity and crystal structure of mutant protein will be studied by the site-directed mutagenesis and protein crystallization. The complementary strains which have been mutated on key amino acid of luxS protein will be constructed, and the signal molecule AI-2 production, biofilm formation, virulence and expression virulence factors will be evaluated. This project will help us to deeply understand LuxS/AI-2 quorum sensing system and provide the theory basis for its regulation mechanism.

细菌luxS基因参与信号分子AI-2合成，LuxS/AI-2型密度感应系统参与调控细菌生物被膜形成、毒力等众多生理功能。本项目组前期通过高效表达猪链球菌LuxS蛋白，获得蛋白晶体，解析了其晶体结构，通过与其他菌属该蛋白一级序列和三维结构叠加比对，发现该蛋白酶底物结合附近第80和87位氨基酸发生了突变，提示该位点的突变可能和LuxS蛋白的进化有关，并可能参与对细菌其他生理功能的调控。本研究以蛋白一级序列和晶体结构为切入点，拟通过定点突变和蛋白结晶技术，体外研究突变蛋白酶活常数和晶体结构变化，并构建可能进化位点氨基酸突变的luxS互补株，探讨突变互补株信号分子AI-2的产生和对生物被膜形成及毒力等的调控作用。本研究旨在解析猪链球菌LuxS蛋白微进化过程中蛋白晶体结构的变化以及重要氨基酸突变对细菌生物被膜形成和毒力等的调控作用，为深入了解LuxS/AI-2型密度感应系统及其调控机理提供理论依据。

细菌luxS基因参与信号分子AI-2合成，LuxS/AI-2型密度感应系统参与调控细菌生物被膜形成、毒力等众多生理功能。本项目组通过高效表达猪链球菌LuxS蛋白，获得蛋白晶体，解析了其晶体结构，通过与其他菌属该蛋白一级序列和三维结构叠加比对，发现该蛋白酶底物结合附近第80和87位氨基酸发生了突变，提示该位点的突变可能和LuxS蛋白的进化有关，并可能参与对细菌其他生理功能的调控。实验通过定点突变技术对第80和87位氨基酸分别单突变和同时双突变，体外酶学分析发现突变体底物结合能力和催化能力都有一定降低。并将可能的进化位点氨基酸在luxS互补质粒上也进行相同的定点突变，将其电转入luxS缺失株中，构建含有定点突变的luxS互补株，同时研究各菌株产生信号分子AI-2和对生物被膜形成的影响，均发现出现不同程度的影响。本研究解析了猪链球菌LuxS蛋白微进化过程中蛋白晶体结构的变化以及重要氨基酸突变对细菌生物被膜形成和毒力等的调控作用，为深入了解LuxS/AI-2型密度感应系统及其调控机理提供理论依据。