SPD1672蛋白的关键功能域鉴定及其对不同血清型肺炎链球菌合成磷壁酸和毒力的影响研究

发布时间：2018-05-17 16:05

本文选题：肺炎链球菌 + 磷壁酸　；参考：《重庆医科大学》2012年硕士论文

【摘要】：目的磷壁酸(TeichoicAcids)是革兰阳性细菌细胞壁上一种的多糖聚合物，是细菌表面重要的抗原[1]。spd1672基因是本课题组前期筛选出的一个体内诱导基因，生物信息学分析显示该基因在各种血清型肺炎链球菌中的高度保守性，其编码产物具有O抗原连接酶、聚合酶结构域。前期实验结果显示SPD1672蛋白影响了2型肺炎链球菌D39菌株的毒力和磷壁酸合成，可能为磷壁酸合成酶，但其关键结构域尚不清楚。因此本课题拟鉴定SPD1672蛋白影响磷壁酸的关键结构域，并观察其在其它血清型肺炎链球菌中是否也影响到磷壁酸合成及毒力。方法首先通过替代失活的方法构建肺炎链球菌D39（serotype2）、R6(serotype2)、203（serotype3）、TIGR4（serotype4）的spd1672基因缺陷菌株D39△1672、R6△1672、203△1672、TIGR4△1672，利用pEVP3质粒构建以上各菌株的周质环external loop4（EL4）缺陷菌株和spd1672基因的回复菌株，通过突变PCR技术构建5个周质环点突变回复菌株，通过western blot检测野生菌与缺陷菌的各亚组分中磷壁酸合成的差异，鉴定SPD1672蛋白的酶活性位点，并分析其在不同菌株中对壁磷壁酸（WTA）与脂磷壁酸（LTA）合成的影响；采用PJWV25及PAE03质粒荧光报告系统，对SPD1672蛋白进行亚细胞定位分析；通过小鼠生存率的比较、体内定植实验等的毒力实验分析spd1672基因对不同血清型肺炎链球菌毒力的影响。结果通过western blot分析发现， D39菌株缺陷周质环EL4后的磷壁酸合成减少，与缺陷spd1672基因后相一致，说明EL4是其关键功能域；进一步的酶活性位点分析实验发现，第206、270、287、306位氨基酸点突变后磷壁酸的合成量同野生菌相比几乎没有差异，而第304位组氨酸发生点突变后，，其磷壁酸的合成量同缺陷spd1672基因后相一致，较野生菌显著下降，提示该位点为SPD1672蛋白的关键氨基酸；选择抗磷酸胆碱抗体对野生菌及各点突变菌株进行的磷酸胆碱含量分析显示，各突变株的磷酸胆碱变化与磷壁酸含量的变化一致，进一步证实上述结论的可靠性。spd1672基因的N末端和C末端分别与GFP融合表达后，肺炎链球菌的边缘发出荧光，提示该基因表达于细胞膜上。对3型菌株203和4型菌株TIGR4的磷壁酸合成检测结果显示，当缺陷spd1672基因后，缺陷菌全菌组分及各亚组分磷壁酸的合成量都显著减少，说明spd1672基因在这两种血清型菌株中也影响磷壁酸的合成；通过鼻腔的途径感染小鼠，结果发现D39△1672、203△1672、TIGR4△1672同野生菌相比，其在血、鼻咽部、肺及脑中的定植量较野生菌显著减少，缺陷组的小鼠生存率显著高于相应的野生组的小鼠生存率。结论本研究结果提示SPD1672蛋白表达于细胞膜，周质环EL4是其重要的结构功能域，第304位组氨酸是维持其聚合酶活性的关键氨基酸。spd1672基因对2型、3型等血清型菌株的磷壁酸合成都有影响，且影响这些菌株在宿主体内的定植及毒力，提示该基因对肺炎链球菌磷壁酸合成及毒力的影响具有普遍性。
[Abstract]:objective
Phosphoric acid (TeichoicAcids) is a polysaccharide polymer on the cell wall of Gram-positive bacteria. It is an important antigen [1].spd1672 gene on the surface of bacteria. The bioinformatics analysis shows that the gene is highly conserved in a variety of serotype Streptococcus pneumoniae. O antigen ligase, polymerase structure domain. Preliminary results show that SPD1672 protein affects the virulence and phosphoric acid synthesis of Streptococcus pneumoniae strain 2, which may be phosphoric acid synthetase, but its key domain is not clear. Therefore, this topic intends to identify the key domain of SPD1672 protein to influence the phosphoric acid acid, and to observe it in other serum. Whether Streptococcus pneumoniae affects the synthesis and virulence of phosphonic acid, too.
Method
First, by replacing the inactivation of Streptococcus pneumoniae D39 (serotype2), R6 (serotype2), 203 (serotype3), TIGR4 (serotype4), spd1672 gene defective strains D39 delta 1672, R6 Delta 1672203 delta 1672, TIGR4 delta 1672. The mutation PCR technique was used to construct 5 cycle point mutation response strains, and the difference of phosphic acid synthesis in the subcomponents of wild and defective bacteria was detected by Western blot, and the enzyme activity sites of SPD1672 protein were identified, and the effects on the synthesis of pariphiic acid (WTA) and lipophosphoric acid (LTA) in different strains were analyzed. PJWV25 and PAE03 were used. The plasmid fluorescence reporting system was used for subcellular localization of SPD1672 protein, and the effect of spd1672 gene on the virulence of different serotypes of Streptococcus pneumoniae was analyzed by comparison of the survival rate of mice and in vivo colonization experiments.
Result
The Western blot analysis showed that the phosphorus wall acid synthesis decreased after the defective cycle EL4 of the D39 strain, which was consistent with the defective spd1672 gene, indicating that EL4 was the key functional domain. Further enzyme active site analysis showed that the synthesis of phosphoric acid acid was almost no worse than that of wild bacteria after 206270287306th amino acid sites mutation. After the mutation of the 304th - bit histidine point, the synthesis of phosphoric acid was consistent with the defective spd1672 gene, which was significantly lower than that of the wild bacteria, suggesting that the site was the key amino acid of SPD1672 protein; the phosphoric acid choline content analysis of wild and point mutant strains selected by anti phosphaticholine antibody showed that the phosphorus of each mutant strain was found. The changes in acid choline were consistent with the changes in the content of phosphoric acid acid. It was further confirmed that the N and C ends of the.Spd1672 gene were fused with GFP, and the edge of Streptococcus pneumoniae emitted fluorescence, suggesting that the gene was expressed on the cell membrane. The results of the phosphoric acid synthesis detection for the 203 and 4 strains of type 3 strain TIGR4 showed that, After the defective spd1672 gene, the total and subcomponents of the defective bacteria were significantly reduced, indicating that the spd1672 gene was also affected by the synthesis of phosphoric acid in these two serotype strains. The results showed that the D39 Delta 1672203 delta 1672 and the TIGR4 delta 1672 were in the blood, nasopharynx, and lung compared with the wild bacteria through the nasal cavity. The survival rate of mice in the defect group was significantly higher than that in the wild group.
conclusion
The results of this study suggest that SPD1672 protein is expressed in the cell membrane, and the periplasmic ring EL4 is an important structural functional domain. 304th - bit histidine is the key amino acid.Spd1672 gene for maintaining its polymerase activity, which affects the phosphoric acid acid synthase of type 2 and type 3 serotype strains, and affects the colonization and virulence of these strains in the host. The effect of genes on the synthesis and virulence of pneumococcal Streptococcus pneumoniae is universal.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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