肺炎链球菌假想蛋白SPD0873功能分析及对细菌毒力影响的机制研究

发布时间：2018-03-30 16:03

本文选题：肺炎链球菌　切入点：假想蛋白　出处：《重庆医科大学》2010年硕士论文

【摘要】： 背景由于肺炎链球菌(Streptococcus pneumoniae,S.pn)抗生素耐药率的增加和目前疫苗本身的缺陷,使得其防治成为了一个很棘手的问题。要想彻底解决这个问题,就要深入其致病机制,为新药物和疫苗的开发提供理论依据。在前期研究中,我们通过体内表达技术(IVET),筛选出了与肺炎链球菌毒力相关的体内诱导基因(in vivo-induced gene, ivi gene)。经过对这些基因的生物信息学分析,我们发现spd-0873基因所编码的产物,假想蛋白SPD-0873与肺炎链球菌中的LytC有相似的功能结构域,LytC的阴性突变菌株生物被膜形成能力下降。所以我们推测它是一个功能类似于LytC的新的毒力因子。目的本研究将对假想蛋白SPD-0873的生物学功能进行鉴定,并探索spd-0873基因影响细菌毒力的机制。方法通过水解底物PNP-(GlcNAc)5实验来确定蛋白SPD-0873是否具有溶菌酶的功能;采用长臂同源多聚酶链式反应(Long flanking homology polymerase chain reaction,LFH-PCR)方法制备spd-0873缺陷菌,利用小鼠体内实验比较缺陷菌和野生菌的毒力是否存在差异;通过描绘细菌生长曲线、透射电镜、荚膜染色来观察体内、外荚膜合成能力、体外粘附宿主细胞实验等方法,进一步了解假想蛋白SPD-0873的功能,通过荧光定量PCR比较缺陷菌和野生菌中几种主要的毒力因子的表达差异,探索spd-0873基因影响细菌毒力的机制。结果通过生物信息学分析可知, spd-0873的编码产物含有β-1,4-N-乙酰胞壁酸糖苷酶结构域,该酶属于糖基水解酶25家族,可以水解细胞壁中N-乙酰胞壁酸(NAM)和N-乙酰葡糖按(NAG)之间的β-1,4糖苷键。溶菌酶实验结果显示,卵清溶菌酶和SPD-0873重组蛋白均可使底物PNP-(GlcNAc)5释放出游离的对硝基苯酚,OD405nm吸光度值分别为1.166和0.792;小鼠体内毒力实验显示,野生菌和缺陷菌的死亡时间分别是感染后11h和36h,缺陷菌毒力较野生菌显著降低(P0.001);体外生长曲线显示,缺陷菌的增殖能力较之野生菌显著降低;透射电镜显示:体外培养缺陷菌荚膜丢失,感染小鼠后荚膜可恢复,而野生菌体外、体内均有荚膜;缺陷菌体外粘附能力较之野生菌显著增强(P0.01);同野生菌比较,溶血素(pneumolysin, ply)、神经氨酸酶(neuraminidase, nanA)在缺陷菌中表达量分别降低了1.2倍、1.26倍,而链球菌表面粘附素A(pneumococcal surface adhesion A, psaA)表达量增加3倍。结论实验结果提示假想蛋白SPD-0873具有一定的溶菌酶活性;同时spd-0873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生长增殖、荚膜形成、粘附能力及毒力因子的表达,是一种新的毒力因子。
[Abstract]:Background because of the increase of antibiotic resistance rate of Streptococcus pneumoniae S.pn) and the defect of the vaccine itself, the prevention and treatment of Streptococcus pneumoniae S.pn. has become a very difficult problem.In order to solve this problem thoroughly, we should go deep into its pathogenic mechanism and provide theoretical basis for the development of new drugs and vaccines.In previous studies, we screened out in vivo vivo-induced gene and ivi gene associated with virulence of Streptococcus pneumoniae by in vivo expression technique.Through bioinformatics analysis of these genes, we found that the product encoded by spd-0873 gene, the hypothetical protein SPD-0873 and the LytC in Streptococcus pneumoniae, had similar functional domain, and the biofilm formation ability of the negative mutant strain LytC was decreased.So we speculate that it is a new virulence factor that functions like LytC.Objective to identify the biological function of hypothetical protein SPD-0873 and explore the mechanism of spd-0873 gene affecting bacterial virulence.Methods PNP-(GlcNAc)5 assay was used to determine whether the protein SPD-0873 had lysozyme function, long flanking homology polymerase chain reaction was used to prepare spd-0873 deficient bacteria.The virulence of defective bacteria was compared with that of wild bacteria in mice, and the ability of capsule synthesis in vivo and in vitro was observed by describing the growth curve of bacteria, transmission electron microscope, capsule staining, and so on.To further understand the function of hypothetical protein SPD-0873, the mechanism of spd-0873 gene affecting bacterial virulence was explored by comparing the expression differences of several main virulence factors between defective bacteria and wild bacteria by fluorescence quantitative PCR.Results according to the bioinformatics analysis, the encoding product of spd-0873 contained 尾 -1C 4-N acetylcyticosidase domain, which belonged to the glycosylhydrolase 25 family.The 尾 -1C 4 glucoside bond between N- acetyl cytosolic acid (NAM) and N-acetyl glucosamine (NAG) in cell wall can be hydrolyzed.The death time of wild bacteria and deficient bacteria were 11 h and 36 h after infection respectively, the virulence of defective bacteria was significantly lower than that of wild bacteria, and the growth curve in vitro showed that the proliferative ability of defective bacteria was significantly lower than that of wild bacteria.Transmission electron microscopy (TEM) showed that the capsule of defective bacteria was lost in vitro, the capsule recovered after infection in mice, while the capsule of wild bacteria was found in vivo, the adhesion ability of defective bacteria in vitro was significantly enhanced than that of wild bacteria, and compared with wild bacteria, the in vitro adhesion of defective bacteria was significantly higher than that of wild bacteria, while that of wild bacteria was higher than that of wild bacteria.The expression of hemolysin, plyosin, neuraminidase and neuraminidase were decreased by 1.2-fold and 1.26 times, respectively, while the expression of A(pneumococcal surface adhesion A (psaA-1) increased by three times.Conclusion the results suggest that the hypothetical protein SPD-0873 has a certain lysozyme activity, and that the virulence of spd-0873 gene deficient bacteria in mice is significantly decreased, and the growth and proliferation of Streptococcus pneumoniae, capsule formation, adhesion ability and the expression of virulence factor are also affected.It is a new virulence factor.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.14

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