【作者】 牛司强；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2011， 博士

【摘要】 由于抗生素耐药的增加和疫苗本身存在的缺陷,肺炎链球菌的预防和治疗面临着很大的挑战。这就需要我们进一步深入研究肺炎链球菌的致病机理,为抗菌药物的开发和疫苗的研制提供理论基础。前期工作中课题组研究发现,肺炎链球菌的一个体内诱导基因sp0987编码的假想蛋白质SP0987是一个潜在的毒力因子。到目前为止,SP0987的结构和功能都不清楚,需要我们做进一步的研究。目的本研究将用X-射线晶体学方法首次解析假想蛋白质SP0987的三维立体结构,在结构的基础上研究其生物学功能,并探索sp0987基因影响细菌毒力的初步机制。方法通过克隆、表达、纯化、蛋白质晶体培养、衍射数据收集和蛋白质三维立体结构解析,得到SP0987的三维立体结构;从三维结构所提供的信息,运用酶活性分析方法和定点突变技术进行溶菌酶活性的分析和酶活性位点的分析与验证;通过荧光报告系统来确定SP0987的细胞定位;通过构建肺炎链球菌sp0987缺陷菌株研究小鼠生存实验及体内的定植实验和体外的细胞粘附和侵袭实验,来研究sp0987基因缺陷后对肺炎链球菌致病性的影响。结果生物信息学分析显示,由基因sp0987编码的假想蛋白质SP0987全长266个氨基酸,其编码产物可能为溶菌酶,主要作用是降解细胞壁中N-乙酰胞壁酸和N-乙酰葡糖胺之间的β-1,4-糖苷键,属于GH25家族溶菌酶;通过克隆、表达、纯化、蛋白质晶体培养、衍射数据收集和蛋白质三维立体结构解析,首次成功解析SP0987的三维立体结构;序列比对和三维结构叠合分析发现,SP0987是溶菌酶样蛋白质,保守性分析及结构特点分析发现可能的活性位点D33、D131、E133和D222,且都为酸性氨基酸残基;分子筛实验提示,SP0987在溶液中以单体形式存在;应用绿色荧光蛋白报告系统分析SP0987的细胞定位,结果发现SP0987定位于细胞膜,与生物信息学分析结果一致;用LFH-PCR技术构建sp0987缺陷菌,小鼠体内生存实验结果显示,sp0987缺陷后肺炎链球菌毒力明显减弱;体外细胞实验发现,sp0987缺陷菌对宿主细胞的侵袭明显弱于野生菌,而对宿主细胞的粘附没有明显差异;体内定植实验发现,肺炎链球菌D39野生菌及缺陷菌经鼻腔感染后,缺陷菌入血时间明显较晚,且在BALB/c小鼠鼻咽部和肺部的细菌载量有明显差异,缺陷菌的细菌载量明显减少。结论用X-射线晶体学方法首次成功解析肺炎链球菌假想蛋白质SP0987的三维结构;结构分析及功能实验发现,SP0987是溶菌酶样膜蛋白,在溶液中以单体形式存在;毒力实验显示,sp0987基因缺陷菌在小鼠体内毒力显著降低,并影响肺炎链球菌的侵袭能力和定植能力,所以其编码产物SP0987是一种新的毒力因子。更多还原

【Abstract】 Streptococcus pneumoniae is a serious challenge for the prevention and therapy because of the increasing antibiotic resistance and the defects of the current vaccine. So further research should be done on the pathogenic mechanism of Streptococcus pneumoniae, which can supply the theory background of more effective antibiotic and vaccine development. In our previous study, we have found that a putative protein SP0987 from Streptococcus pneumoniae coded by an in vivo induction gene sp0987, may be a new virulence factor. Till now, the structure and function of SP0987 was unknown, so we decided to do further research on the protein.Objectives The aim of this study was to resole the structure of the protein SP0987 for the first time, to study the function based on the structure and the effect of the sp0987 gene on the pathogenesis of Streptococcus pneumoniae.Methods The structure of sp0987 was analysed by clone, expression, purification, protein crystal growth, diffraction data collection and protein structure resolution. Based on the structure, the enzyme activity and the active sites were analysed through the analysis of enzyme activity and site-directed mutagenesis. The cellular localization of SP0987 was analysed using novel fluorescent reporter system.The virulence study was carried out by the construction of the sp0987 mutant strain, cell adherence and invasion experiment in vitro, colonization experiment in vivo.Results The putative protein SP0987 , coded by the gene sp0987, 266 amino acid, was predicted by bioinformatics to be aβ-1,4-N-acetylmuramidase belonging to glycosylhydrolase family 25, which cleaved theβ-1,4-glycosidic bond between N-acetylmuramic acid(NAM) and N-acetylglucosamine(NAG) in the carbohydrate backbone of bacterial peptidoglycan. The structure of SP0987 was resolved successfully for the first time by clone, expression, purification, protein crystal growth, diffraction data collection and protein structure resolution. By sequence and structural comparison of SP0987 with other lysozymes, we found that SP0987 was a lysozyme-like protein, and the active sites were probably D33, D131, E133 and D222. The protein SP0987 was proved to be a monomer state in solution by size exclusion chromatography; A GFP fusion to SP0987 showed clear membrane localization, as predicted from its primary sequence. The sp0987 gene mutant stain was constructed by LFH-PCR, and the reduced virulence of the mutant suggested the SP0987 may be a virulence factor in Streptococcus pneumoniae. In vitro, the invasion rate of the mutant strain to the host cell was lower than the wild strain, while there was no difference between the mutant stain and the wild strain in adherence. In vivo, when mice were challenged intranazal, the wild strain appeared in blood earlier than the mutant strain, and the number of bacteria in the nasal and lung tissue of the mutant was much smaller than the wild strain.Conclusions The structure of the putative protein SP0987 from Streptococcus pneumoniae was resolved for the first time by X-ray crystallography. The protein SP0987 was a new lysozyme-like membrane protein, and was a monomer state in solution. The sp0987 gene mutant strain showed reduced virulence in the intranasal mouse model , and affected invasion and colonization of Streptococcus pneumoniae. So, the protein SP0987 is a new virulence factor of Streptococcus pneumoniae.更多还原