【作者】 张群；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2009， 博士

【摘要】 肺炎链球菌(Streptococcus pneumoniae, S.pn)是一种常见的革兰阳性条件致病菌,可引起肺炎、中耳炎、菌血症、脑膜炎等严重疾病,在全球有很高的发病率和死亡率。老人、儿童、艾滋病和先天性免疫缺陷的患者是S.pn感染的高危人群。由于S.pn抗生素耐药率的增加和目前疫苗的缺陷性,使得其防治面临困境。要想从根本上解决S.pn的防治问题,就要深入了解其致病的分子机制,为研发新的药物和疫苗提供理论和实验依据。HSP100/ ClpATPase是一类高度保守并广泛存在的热休克蛋白(heat shock protein, HSP),它除了具有分子伴侣的功能外,还与肽酶ClpP形成ATP依赖的蛋白酶复合物(如ClpAP,ClpBP等)特异性水解某些蛋白质。研究显示ClpATPase可影响某些基因的转录因子活性,从而调控包括毒力因子在内的多种基因的表达,导致细菌生长、毒力等多种生物学性状发生改变。肺炎链球菌有四种ClpATPase(ClpC,ClpE,ClpL,ClpX)。已有的研究显示ClpC、ClpL及ClpX都与S.pn的感染致病相关。但ClpE目前仅知道是肺炎链球菌温度耐受的主要元件,对于其在肺炎链球菌致病过程中的作用尚不清楚。本课题从整体水平、细胞水平和分子水平初步探索了ClpE在肺炎链球菌致病过程中的作用及机制,这可为深入了解肺炎链球菌致病机制提供理论和实验依据。本课题包括以下两个部分内容:1、构建肺炎链球菌clpE缺失突变体,比较clpE缺陷菌株和野生菌D39对小鼠的毒力差异,从总体水平上初步了解ClpE是否参与了肺炎链球菌的致病。采用长臂同源多聚酶链式反应(Long flanking homology polymerase chain reaction,LFH-PCR)方法,制备中间为红霉素耐药基因(erm)两侧为clpE基因上、下游同源序列的连接片段,并将此片段转化入肺炎链球菌D39,在含红霉素的血平板上筛选出clpE缺陷菌株,并通过PCR、测序进行鉴定;小鼠体内实验比较clpE缺陷菌株和野生菌D39对小鼠的毒力差异。PCR和测序结果显示,我们构建D39clpE缺陷菌株成功;小鼠体内实验结果显示,clpE缺陷菌株对小鼠的毒力较野生菌株明显减低(P<0.01)。研究结果提示,ClpE可能在肺炎链球菌的致病过程中发挥了作用。2、从细胞水平、分子水平进一步了解ClpE在肺炎链球菌致病过程中的作用及其机制。本研究观察了不同温度下clpE缺陷菌株和野生菌株在液体培养基中的生长情况;体外粘附、侵袭实验比较缺陷菌株与野生菌株对宿主细胞(人肺腺癌细胞A549、人脐静脉内皮细胞HUVEC)的粘附、侵袭能力;荧光定量PCR检测肺炎链球菌几种主要毒力因子在缺陷菌株和野生菌株中的表达差异;双向凝胶电泳比较缺陷菌株和野生菌株的蛋白表达差异,借助基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对部分蛋白质分子进行分析鉴定,并结合生物信息学方法寻找ClpE调节的蛋白质分子。结果显示:1、在37℃时,clpE缺陷菌的生长繁殖能力较野生菌株大大降低,而在40℃、42℃时,缺陷菌几乎不能生长。2、clpE缺陷菌株对宿主细胞的粘附和侵袭能力都显著弱于相应的野生菌株(P<0.05)。3、同野生菌株比较,几种重要毒力基因自溶素(major autolysin A,lytA)、表面粘附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、神经酰胺酶(neuraminidase, nanA)和链球菌表面蛋白A(pneumococcal surface protein A, pspA)在clpE缺陷菌中的表达量显著下降(P<0.05)。4、与细菌生长、粘附侵袭、生物被膜形成密切相关的蛋白质如次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine-guanine phosphoribosyltransferase)、甲酸-四氢叶酸酯连接酶( Formate-tetrahydrofolate ligase )、吡咯烷羧酸肽酶(Pyrrolidone-carboxylate peptidase 1)和双功能蛋白PyrR (bifunctional protein PyrR)在缺陷菌中的表达量均明显低于其野生菌。这些结果提示ClpE可能通过调控包括毒力因子在内的一些蛋白质的表达来影响肺炎链球菌的生存、对宿主细胞粘附、侵袭等多种生物学功能。总之,本研究显示ClpE可通过调控多种蛋白质的表达来影响细菌的生存能力、对宿主细胞的粘附侵袭能力以及对宿主细胞的损伤能力,它是肺炎链球菌在宿主体内存活并导致感染发生所必须的。更多还原

【Abstract】 Among the gram-positive opportunistic human pathogens, Streptococcus pneumoniae is a major cause of serious invasive diseases, including pneumonia, otitis media, bacteremia, and meningitis. It remains the high morbidity and mortality throughout the world, particularly in infants, elders, and immunocompromised patients. S. pneumoniae infection is a widespread serious problem because of the increasing antibiotic resistance and defects of the current vaccine. Therefore, we should further understand its molecular pathogenic mechanism to provide new theory and experimental data for more effective drug and vaccine development.HSP100/ ClpATPase ( Heat shock proteins 100 ) are extremely conserved in prokaryotes. Members of the HSP100 family represent proteins that form the largest chaperone and protease complexes in bacterial cells. Previous studies have revealed that ClpATPase are important for not only cell physiology but also regulation of virulence properties of pathogenic bacteria. In Streptococcus pneumoniae, Clp ATP-dependent proteases were composed of a proteolytic subunit, ClpP, and an ATPase subunit (ClpC, ClpE, ClpL, and ClpX) accounting for substrate specificity. ClpC ATPase is the interacting partner of ClpP in many physiologically important processes such as pneumolysin release and adaptation to diverse stress parameters. ClpL not only modulates the virulence gene expression but also affects the adherence and invasion to host cells. Furthermore, in S. pneumoniae R6, depletion of ClpX leads to rapid cell death without overtly affecting cell morphology. Although ClpE of S.pn has been shown to be required for growth at high temperature, the role of ClpE on pathogenesis has not been described so far. The purpose of this study was to investigate the effect of ClpE on the pathogenesis of Streptococcus pneumoniae. This study includes the following two parts:1. Construction of clpE-deletion mutant of S.pneumoniae and investigation of the effect of ClpE on the pathogenesis of Streptococcus pneumoniae.LFH-PCR (Long flanking homology polymerase chain reaction) was introduced to generate a gene disruption construct consisting of erm cassette with long flanking homology regions to the target gene. Then S.pneumoniae D39 was transformed directly with this PCR product. The clpE-deletion mutant was obtained on the TSA agar containing erythromycin and identified by PCR and sequencing. The impact of clpE mutant on the virulence of S. pneumoniae was evaluated in a mouse intraperitoneal challenge model. Our results showed that clpE gene was completely replaced by erm cassette. The virulence of clpE mutant was significantly attenuated in mice intraperitoneal infection model. The results implicated that ClpE was involved in the pathopoiesis of Streptococcus pneumoniae.2. Investigation of the role of ClpE in the pathopoiesis of Streptococcus pneumoniae.Growth of parental strain (D39) and clpE-deletion strain at different temperatures were examined in C+Y media. In addition, we investigated the effect of clpE mutant on adherence and invasion to host cells (human lung epithelial carcinoma A549 cells line and human umbilical vein-derived endothelial cells HUVEC). The underlying molecular mechanism of virulence attenuation induced by the mutation of clpE was further investigated by FQ-PCR (fluorescent quantitative polymerase chain reaction) and two-dimensional protein gel analysis.The growth of clpE mutant was slower than that of the parent at 37°C. The clpE mutants presented a temperature-sensitive growth phenotype at 40°C and 42°C. Cell culture infection experiments indicated that adherence and invasion of isogenic mutants in which the clpE gene was inactivated were both strongly reduced. FQ-PCR analysis demonstrated that the expression of major virulence determinants, such as pneumolysin (ply), pneumococcal surface antigen A (psaA), neuraminidase (nanA), pneumococcal surface protein A (pspA) and major autolysin (lytA) were decreased in clpE mutants. The protein expression analysis led to the identification of hypoxanthine–guanine phosphoribosyltransferase (HPRT), pyrrolidone-carboxylate peptidase 1, formate-tetrahydrofolate ligase, and bifunctional protein PyrR, which were down regulated in the D39ΔclpE and involved in the survival and adhesion of bacteria. These results indicated that ClpE could help S.pneumonia invading and adapting to different environments of the host by participating in the expression of special proteins mentioned above.In a word, ClpE is required for the ability of bacterial surviving, adhering and invading to host cells, and it plays an important role in the pathopoiesis of Streptococcus pneumoniae.更多还原