猪链球菌2型是重要的人兽共患病原菌，临床菌株多呈严重耐药和多重耐药，应用抗生素治疗面临挑战。猪链球菌2型噬菌体编码的裂解酶LySMP因呈现高效裂菌活性而具有抗菌应用潜力，但其介导裂菌的酶学基础和裂菌机制尚不清楚。本项目将聚焦LySMP的功能域解析和底物识别机制研究：通过生物信息学、定位突变和负离子质谱分析，解析LySMP的催化域和结合域，揭示酶活性中心、酶切作用靶点；应用免疫共沉淀和质谱分析，发掘猪链球菌细胞壁上的LySMP结合域受体；利用等温滴定量热法（ITC)和表面等离子共振（SPR）技术，测定LySMP的底物结合常数和催化效率，阐明酶学特征。采用蛋白质结晶、X射线衍射和同源建模解析裂解酶的三维结构，并基于核磁共振技术重建猪链球菌肽聚糖的三维结构，进而揭示裂解酶-肽聚糖复合物的动态三维结构特征，最终阐明LySMP的底物识别和裂解机制，为研制防控猪链球菌病的高效裂解酶奠定理论和应用基础。

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen, and the increasing prevalence of antibiotics resistant SS2 strains becomes one of the greatest challenges worldwide. LySMP, a peptidoglycan hydrolase （lysin） encoded by SS2 bacteriophage, has been examined as potential agent for SS2 control, but its enzyme kinetics and bacteriolytic molecular mechanism are still open. This proposal will focus on the revelation of three-dimensional (3D) structure of LySMP and the elucidation of molecular recognition of bacterial peptidoglycan by LySMP. So, the characteristics of catalytic domain and binding domain of LySMP, including the catalytic active site residues and even the cleaved bonds in peptidoglycan will be investigated by using bioinformatics, site-directed mutation and negative ion mass spectrum. The receptor of LySMP binding domain will be characterized by co-immunoprecipitation and mass spectrum. The LySMP binding constants and catalytic efficiency will be measured by using a combination of isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) in conjunction with active site mutants and deletion constructs. The 3D structure of LySMP will be reconstructed by crystallization and X-ray diffraction or homology modeling, and the 3D structure of peptidoglycan or LySMP-peptidoglycan complex will be solved by nuclear magnetic resonance( NMR) spectroscopy. These findings will allow the mechanism to be revealed on how LySMP recognizes and lyses the bacterial cell wall, which is crucial to improve and reconstruct the LySMP for the control of SS2 infection.