一种肺炎链球菌体磷壁酸合成相关蛋白SPD1672功能研究

发布时间：2018-06-22 15:35

本文选题：肺炎链球菌 + D39Δ1672　；参考：《重庆医科大学》2011年硕士论文

【摘要】：背景本课题组前期工作从肺炎链球菌中筛选到一些体内诱导基因,这些体内诱导基因可能与细菌感染致病有关。spd1672基因是其中的一个体内诱导基因,经过生物信息学分析,其表达产物SPD1672蛋白含有脂质A核心-O抗原连接酶结构域,提示该基因可能与细菌细胞壁多糖形成相关,本课题拟对spd1672基因功能进行鉴定,探寻它影响细胞壁多糖合成的生物学证据,并进一步研究其在感染致病过程中的毒力机制。方法首先通过基因替代失活法构建肺炎链球菌spd1672基因缺陷菌(D39△1672);通过透射电子显微镜对细菌荚膜厚度进行观察;通过C反应蛋白结合实验及补体C3沉积实验观察细菌磷壁酸合成量改变,并进一步对细胞壁原生质及细胞壁壁进行分离,通过western blot确证野生菌与缺陷菌壁磷壁酸(WTA)及脂磷壁酸(LTA)合成量变化情况。通过增加Mg~(2+)浓度改变培养基渗透压,观察野生菌与缺陷菌对高渗环境的耐受能力。用小鼠毒力实验比较spd1672缺陷菌与野生菌的毒力改变;并通过体内定植实验、杀菌实验、细胞因子测定等研究方法,对spd1672影响肺炎链球菌毒力的机制进行初步探讨。结果D39△1672较D39在体外培养条件下生长能力减弱,通过透视电子显微镜观察,D39△1672与D39荚膜厚度并无明显改变。C反应蛋白结合实验结果显示D39△1 672结合CRP量较D39明显减少。而补体C3沉积实验结果显示D39△1672结合补体C3量较D39明显减少。进一步利用抗磷壁酸抗体对两种细菌磷壁酸合成量进行western blot分析后发现,D39△1672较D39菌WTA和LTA合成量菌明显减少。改变生长培养基的离子浓度后发现,在加入50mM Mg~(2+)条件下,D39△1672的增殖速率较正常培养基中明显提高(P0.05),而对D39并无明显影响,而在100mM、200mM Mg~(2+)条件下,D39△1672的增殖受到显著抑制(P0.05),而D39仅受轻微影响(P0.05)。通过腹腔途径感染小鼠,结果显示D39△1672毒力较D39菌明显减弱,其感染小鼠后入血菌量较D39明显减少(P0.05),在鼻咽部、肺部定植数也较D39显著减少(P0.05)。全血杀菌实验结果显示D39△1 672对白细胞的杀菌作用抵抗力较D39显著减弱(P0.05)。通过对细胞因子测定,结果发现D39感染的小鼠较D39△1 672诱导更高水平的IL12、TNF-α。结论通过本研究显示spd1672基因是影响磷壁酸合成的关键基因,该基因缺失后,会引起细菌磷壁酸合成显著减少。同时该基因缺陷后,细菌生长增殖能力减弱;对宿主定植、侵袭能力减弱;对白细胞杀菌效应的抵抗力减弱;并对Mg~(2+)渗透压等外界环境改变更为敏感,提示该基因是细菌感染致病过程中的一个重要的毒力因子。
[Abstract]:Background in our earlier work, some induced genes were screened from Streptococcus pneumoniae. These induced genes may be related to bacterial infection. SPD1672 gene is one of them, which is analyzed by bioinformatics. The expression product SPD1672 contained the ligase domain of lipid A core-O antigen, suggesting that this gene may be related to the formation of bacterial cell wall polysaccharides. The purpose of this study is to identify the function of spd1672 gene. To explore the biological evidence of its influence on cell wall polysaccharide synthesis and to further study its virulence mechanism in the process of infection. Methods spd1672 gene deficient bacteria of Streptococcus pneumoniae (D39 1672) were constructed by the method of gene replacement inactivation, and the thickness of the capsule was observed by transmission electron microscope (TEM). C-reactive protein binding assay and complement C3 deposition were used to observe the changes of phosphatidic acid synthesis, and the protoplasm and cell wall of cell wall were further separated. Western blot was used to confirm the changes of the amount of phosphatidic acid (WTA) and phosphatidic acid (LTA) in the wall of wild and defective bacteria. The tolerance of wild and defective bacteria to hyperosmotic environment was observed by increasing the concentration of Mg2 and changing the osmotic pressure of culture medium. The virulence changes of spd1672 deficient bacteria and wild bacteria were compared with mice virulence test, and the mechanism of spd1672 affecting the virulence of Streptococcus pneumoniae was preliminarily discussed by means of colonization experiment, bactericidal test and cytokine determination in vivo. Results the growth ability of D39 1672 was weaker than that of D39 in vitro. The thickness of D39 1672 and D39 capsule did not change obviously. The results showed that the amount of D39 1 672 binding CRP was significantly lower than that of D39. The results of C _ 3 deposition showed that C _ 3 content of D _ 39 1672 was significantly lower than that of D _ (39). The results of western blot analysis showed that D39 1672 was significantly lower than that of D39 strain. After changing the ion concentration of the growth medium, it was found that the proliferation rate of D39 1672 was significantly higher than that of the normal medium under the condition of 50 mm mg ~ (2) addition (P0.05), but had no obvious effect on D39. The proliferation of D39 1672 was significantly inhibited (P0.05) under 100mM ~ (2) mg ~ (2), while D39 was only slightly affected (P0.05). The results showed that the virulence of D39 1672 was significantly lower than that of D39, and the amount of blood bacteria in infected mice was significantly lower than that of D39 (P0.05), and the number of lung colonization in nasopharynx was significantly lower than that in D39 (P0.05). The results of whole blood germicidal test showed that the bactericidal resistance of D391,672 to leukocytes was significantly lower than that of D39 (P0.05). The results showed that the level of IL-12 TNF- 伪 in D39 infected mice was higher than that in D39 1,672 mice. Conclusion this study shows that spd1672 gene is the key gene affecting the phosphoric acid synthesis, and the deletion of the gene will cause a significant decrease in the phosphoric acid synthesis of bacteria. At the same time, the ability of bacteria growth and proliferation was weakened, the ability of invasiveness to host was weakened, the resistance to leukocyte bactericidal effect was weakened, and was more sensitive to the change of mg ~ (2) osmotic pressure. It is suggested that this gene is an important virulence factor in the pathogenic process of bacterial infection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.14

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