一种肺炎链球菌溶菌酶样假想蛋白质SP0987的结构和功能研究

发布时间：2018-06-07 16:06

本文选题：肺炎链球菌 + 毒力因子　；参考：《重庆医科大学》2011年博士论文

【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。
[Abstract]:The prevention and treatment of Streptococcus pneumoniae is facing great challenge due to the increase of antibiotic resistance and the defects of the vaccine itself. It is necessary for us to further study the pathogenic mechanism of Streptococcus pneumoniae and provide a theoretical basis for the development of antimicrobial agents and vaccines. In previous work, we found that SP0987, a hypothetical protein encoding gene sp0987, is a potential virulence factor of Streptococcus pneumoniae. So far, the structure and function of SP0987 are not clear, so we need further study. Objective to analyze the three-dimensional structure of hypothetical protein SP0987 by X-ray crystallography for the first time, to study its biological function based on the structure, and to explore the mechanism of sp0987 gene affecting bacterial virulence. Methods the three-dimensional structure of SP0987 was obtained by cloning, expression, purification, protein crystal culture, diffraction data collection and protein three-dimensional structure analysis. The lysozyme activity and the site of lysozyme activity were analyzed and verified by enzyme activity analysis and site-directed mutagenesis, and the cellular location of SP0987 was determined by fluorescence report system. In order to study the pathogenicity of streptococcus pneumoniae by constructing a strain of Streptococcus pneumoniae sp0987 deficient in vivo and colonization and in vitro cell adhesion and invasion test the effect of sp0987 gene deficiency on the pathogenicity of Streptococcus pneumoniae was studied. Results Bioinformatics analysis showed that the hypothetical protein SP0987 encoded by gene sp0987 was 266 amino acids in length, and its encoding product might be lysozyme, which was mainly responsible for the degradation of 尾 -1-glucoside bond between N-acetylcytidic acid and N-acetyl glucosamine in cell wall. By cloning, expression, purification, protein crystal culture, diffraction data collection and protein three-dimensional structure analysis, the three-dimensional structure of SP0987 was successfully analyzed for the first time. Sequence alignment and three dimensional structure analysis revealed that SSP0987 was lysozyme like protein. Conserved analysis and structural characteristics analysis showed that the possible active sites were D33OD131E133 and D222, and both were acidic amino acid residues. Molecular sieve experiment indicated that SP0987 existed in the form of monomer in solution, the cell localization of SP0987 was analyzed by green fluorescent protein report system, and the results showed that SP0987 was located on cell membrane, which was consistent with the result of bioinformatics analysis. Sp0987 deficient bacteria were constructed by LFH-PCR technique. In vivo survival test showed that the virulence of Streptococcus pneumoniae was significantly decreased after the deficiency of sp0987, and the invasion of host cells was weaker than that of wild bacteria in vitro, but there was no significant difference in adhesion to host cells. In vivo colonization experiment, it was found that the blood entry time of Streptococcus pneumoniae D39 wild bacteria and defective bacteria was obviously late, and the bacterial load in nasopharynx and lung of BALB/c mice was significantly different, and the bacterial load of defective bacteria was obviously decreased. Conclusion the three-dimensional structure of SP0987 of Streptococcus pneumoniae was successfully elucidated by X-ray crystallography for the first time, and the results of structural analysis and functional experiments showed that SP0987 was a lysozyme-like membrane protein, which existed in the form of monomer in solution. The virulence test showed that the virulence of SNP0987 was significantly decreased in mice, and the invasion and colonization ability of Streptococcus pneumoniae were affected. Therefore, SP0987 was a new virulence factor.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

【参考文献】