| 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.
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