【作者】 胥文春；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2007， 博士

【摘要】 尽管抗生素治疗已取得了长足的进步,细菌性脑膜炎仍是中枢神经系统发生最频繁,最严重的感染性疾病,其比例至少占脑膜炎总数的60%以上。引起细菌性脑膜炎最常见的三种病原菌依次为流感嗜血杆菌、肺炎链球菌和脑膜炎奈瑟菌。目前对于肺炎链球菌(streptococcus pneumoniae,S.pn)引起脑膜炎的机制并未完全清楚,因此发现S.pn致脑膜炎相关的毒力基因,不仅有助于进一步理解其致病机理,还可为新药开发和研发新型蛋白类疫苗提供候选基因。细菌进入宿主体内就如同进入一个生存竞争的“战场”,要在体内生存就必须表达一些使自身毒力增强或对宿主损伤加剧的基因,因此细菌在宿主体内表达的基因往往就是与致病紧密相关的毒力基因。本研究利用小鼠脑膜炎模型和差异荧光诱导技术(differential fluorescence induction,DFI),在体内大规模地筛选鉴定在肺炎链球菌性脑膜炎感染过程中表达或表达增强的基因,这些基因很可能就是肺炎链球菌引起脑膜炎的相关基因,在很大程度上影响着肺炎链球菌通过血脑屏障(blood-brain barrier,BBB)的侵袭力。研究内容包括以下四个部分。1.肺炎链球菌性脑膜炎动物模型的建立。将冻存的TIGR4在新鲜TSA血平板上培养16~18h后,刮取细菌并稀释成三种不同密度的菌液,每种密度分成2份,其中一份加入一定量的透明质酸酶,另一份不加。对小鼠行浅麻醉,用卡介苗注射器将上述菌悬液缓缓逐滴滴至小鼠鼻腔处,待其自动吸入,每只小鼠50μl。每天观察小鼠情况,于感染后24h、48h和72h处死小鼠,取心脏血和一半脑组织匀浆做细菌计数,另一半脑组织用于组织病理学检查。结果表明:①透明质酸酶并不能促进鼻腔滴注TIGR4致小鼠脑膜炎的发生;②小鼠脑膜炎的发生率与鼻腔感染TIGR4剂量有关,3×10~7CFU已足够,但低于该剂量脑膜炎的发生率会大大降低;③小鼠嗜睡、弓背症状与脑组织病理学改变显著相关。模拟S.pn在人类感染途径,通过鼻腔滴注TIGR4成功建立了小鼠脑膜炎模型,并且证实小鼠可出现嗜睡、弓背等脑膜炎典型症状,为下一步研究S.pn致脑膜炎的分子机制奠定了基础。2.肺炎链球菌绿色荧光蛋白报告质粒的构建及评价。DFI是以绿色荧光蛋白(GFP)为报告基因的筛选病原菌体内诱导基因的技术,必须要构建一个可高效报告基因表达的绿色荧光蛋白报告质粒。质粒pGreenTIR含有SD序列和翻译增强子ENH及无启动子的增强型绿色荧光蛋白基因(gfp),将其中的SD-ENH-GFP片段经BamHⅠ酶切后插入含氯霉素抗性基因的自杀质粒pEVP3中,构建了以绿色荧光蛋白为报告基因的自杀质粒pEVP3-SDGFP。为了评价新建质粒pEVP3-SDGFP报告上游基因表达的情况,我们将肺炎链球菌的溶血素基因(pneumolysin, ply)上游约500bp片段插入到该质粒报告基因上游的多克隆位点中,得到质粒pEVP3-SDGFP-Ply,将其转化入肺炎链球菌TIGR4中,由于溶血素基因在体内、外均可表达,通过体内、外实验证实其不但可以报告肺炎链球菌溶血素基因的表达,并且比已有质粒pEGFP-1报告上游基因表达的能力显著增强。表明构建的肺炎链球菌绿色荧光蛋白报告质粒pEVP3-SDGFP可以用于DFI筛选肺炎链球菌脑组织内诱导基因所需的启动子诱捕文库的构建。3.肺炎链球菌启动子诱捕文库的构建与初步分析。用DFI筛选病原菌体内诱导的基因,首先要构建一个包含无启动子序列的报告基因的启动子诱捕文库。以第一部分构建的自杀质粒pEVP3-SDGFP为骨架,将Sau3AI酶切的肺炎链球菌基因组DNA随机片段(200~800bp)克隆到此载体GFP基因上游的BglⅡ位点,将连接产物转化大肠杆菌DH5α,获得了约58 000个重组子,提取质粒,即为含有肺炎链球菌基因组DNA随机酶切片段的质粒库。考虑到DNA片段的插入方向和大小(200~800bp,平均400bp),该质粒库大约覆盖肺炎链球菌基因组全长(2.2Mb)的5倍,插入率达到90%以上,且有较强的随机性,质量较高。将该质粒库转化入肺炎链球菌TIGR4菌株,获得包含约500 000个肺炎链球菌转化子的菌株库。经过体内和体外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告在特定条件下的基因表达,并且可通过流式细胞仪识别、分选。该文库的成功构建为进一步利用差异荧光诱导技术筛选肺炎链球菌脑组织内诱导基因奠定了良好的基础。4.小鼠脑组织中诱导表达的S.pn基因的筛选及生物信息学分析。将构建好的肺炎链球菌菌株库鼻腔滴注感染BALB/c小鼠,出现嗜睡、弓背等脑膜炎症状后取小鼠脑组织,匀浆,流式细胞仪分选(fluorescence-activated cell sorting, FACS)收集脑组织内有荧光的细菌,体外培养一定时间后,再用流式细胞分选术剔除那些在体外也表达荧光的细菌,收集无荧光表达的细菌,得到只在脑组织内发荧光的细菌,经过两轮筛选,最后我们共挑取220个菌落进行进一步分析。为了克隆这些细菌中包含的脑组织内诱导基因的片段,我们应用酶切自连法,即提取这些菌株的染色体DNA,用BamHI酶切,并自身环化,使整合到染色体上的重组自杀质粒脱落下来并重新环化。对插入的随机片段进行测序后进行生物信息学分析,共证实有24个不同的基因片段,在TIGR4全基因组里面,这24个基因片段所处的操纵元结构中,总共包括51个开放阅读框,其中多数为含有多个ORFs的操纵元结构。这些基因包括参与氨基酸转运和代谢、碳水化合物转运和代谢、DNA复制与重组、细胞壁合成、转录调节、离子摄取以及功能未知的基因,其中有些基因的功能已有一些初步的研究,但其在脑膜炎发生过程中的作用并不清楚,如cbpD、pspA、操纵子psa和操纵子dlt;有一些基因功能完全不清楚。进一步分析和阐明这些基因在脑膜炎发生过程中的作用非常重要。更多还原

【Abstract】 Bacterial meningitis is still the most frequent and severest infectious disease despite the treatment of antibiotic. The three main pathogens of bacterial meningitis are hemophilus influenz, streptococcus pneumoniae and diplococcus meningitis. The pathogenesis of pneumococcal meningitis is not well understood. The identification of new virulence factors of pneumococcal meningitis is very important in investigating more effective drug and vaccine.Upon entering the host, many pathogenic organisms involve themselves in a battlefield where the pathogens have to face the defenses of host immune system for survival. Bacteria respond to this change in circumstances by modulating their patterns of gene expression accordingly, downregulating the expressions of some genes that are no longer necessary, and upregulating those that are specifically required for survival in the host. It seemed reasonable that at least some in vivo-induced genes would play critical roles in the survival and pathogenesis, and serve as potential drug targets and vaccine candidates. In the present work, we employed the differential fluorescence induction and a mouse model of meningitis and identified the genes expressed in the process of meningitis due to streptococcus pneumoniae .These genes are probably the virulence factors of meningitis caused by streptococcus pneumoniae and may play important roles in its ability to transverse the blood-brain barrier. The research work included the following four parts.Firstly, the development of an animal model of pneumococcal meningitis. The animal model of pneumococcal meningitis is necessary to identify virulence factors associated with pneumococcal meningitis. TIGR4 was inoculated from frozen stocks onto tryptic soy agar (TSA) plates supplemented with 5% defibrinated rabbit blood and incubated for 16~18h at 37°C. Bacterial growth was scraped from fresh plates and suspended in phosphate-buffered saline (PBS), and the suspensions were diluted in PBS to the appropriate cell density. They were divided into two parts, and added with or without hyaluronidase respectively. BALB/c mice were anesthetized and infected with 50μl of the suspension via intranasal instillation. At 24h, 48 h and 72h postinfection respectively, mice were sacrificed, blood and half of brains were recovered for bacterial enumeration, and the orther half of brain for histopathologic study. It was found that hyaluronidase was not a necessary factor to establish apneumococcal meningitis via intranasal instillation. The occurance rate of meningitis was related to amount of inoculum. The symptoms of mice were correlated to histologic changes in its brain and this indicated that we can judge the occurance of meningitis through by the symptoms of mice.Secondly, the construction and analysis of a suicide plasmid which employed gfp as a reporter gene. DFI relies on the use of a reporter gene, gfp. The plasmid pGreenTIR has a mutated gfp gene and contains an improved translation initiation region (TIR) for prokaryotes, including the translational enhancer (ENH) and the Shine–Dalgarno (SD) regions of phage T7 gene 10, so that synthesis of GFP is enhanced. The SD-ENH-GFP region of plasmid pGreenTIR was cloned into a suicide plasmid pEVP3 which contains a cat gene encoding resistance protein to chloramphenicol, and a suicide plasmid pEVP3-SDGFP was constructed. To evaluate the function of this plasmid, a 500bp fragment of the pneumolysin gene(ply) of TIGR4 was coloned in the upstream of gfp and then was transformed into streptococcus pneumoniae TIGR4. The results indicated that the plasmid pEVP3-SDGFP could report the expression of ply both in vivo and in vitro, and it can be used to construct the promoter-trap library which is needed in DFI.Thirdly, the construction and analysis of promoter-trap library of S.pneumoniae. The promoter-trap library is very important to screen the in vivo-induced genes by DFI. 200 to 800bp Sau3AI fragments of S. pneumoniae TIGR4 DNA were cloned upstream of the promoterless gfp gene in pEVP3-SDGFP. The ligation was then transformed into E. coli DHα. 58 000 recombinants were obtained. Considering insert DNA orientation and insert size, this represents 5 coverages of the 2.2Mb S.pneumoniae genome, 90% of these clones had 200 to 800bp inserts and the library is random. Transformation by this plasmid library yielded 500 000 S. pneumoniae transformants. Analysed by fluorescence microscope and flow cytometry, this library contains both the in vivo-induced gene fragment and in vitro-expressing fragment. The promoter-trap library is suitable for screening genes induced expression in brain of S. pneumoniae.Lastly, screening of genes induced in brain by DFI and bioinformatics analysis. BALB/c mice were infected intranasally with the promoter-trap library according to the method described in first part. When the mice occurred lethargy and hunched appearance, bacteria were harvested from the brain tissues and sorted by flow cytometry, clones with increased fluorescence were used to reinfect animals for enrichment. After cultured about 48h in vitro, sorted by flow cytometry, clones with low fluorescence were collected. 220 clones were analyzed. To clone the induced fragments, each chromosomal DNA was digested with BamHI, self ligated, and transformed into E coli DH5α. All the recombined suicide plasmids were successfully obtained and sequenced, and 24 unique sequences were identified. Bioinformatics analysis showed that there were 51 ORFs in the 24 operons. These genes involve in the process of amino acid transport and metabolism, carbohydrate transport and metabolism, DNA replication and recombination, cell envelope biogenesis, transcription, and anion/cation acquisition, and some of them, the functions are unknown. Among them, cbpD、pspA、operon psa and operon dlt have been shown that they may involved in the pathogenesis of S. pneumoniae. But the roles of them in pneumococcal meningitis are unknown. It is necessary for further research about these genes in pathogenesis of pneumococcal meningitis.更多还原