| The gram-positive opportunistic human pathogen, 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, the elderly, and immunocompromised patients. S.pneumoniae infection is a widespread and serious problem which results from the increasing antibiotic resistance and the defects of the current vaccine. We should further understand its molecular pathogenic mechanism to provide new theory and experimental data for more effective drug and vaccine development. Upon entering the host, many pathogenic organisims find themselves in a battlefield where the pathogen has 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 a critical role in the survival and pathogenesis, and serve as potential drug targets and vaccie candidates. To identify in vivo-induced genes of S. pneumoniae,in this project,a selection system was developed based on the in vivo expression technology(IVET).We employed galU gene which is critical for the capsular polysaccharide biosynthesis as an in vivo reporter to select S.pneumoniae virulence genes that are specifically induced in host. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose and synthesizing capsular polysaccharide, therefore can't survival in the host. This study includes three partments: 1.Construction and function study of galU-deletion mutant in S.pneumoniae To construct galU-deletion mutant of S.pneumoniae and get the capsule deficient strain which can be used in following research of ivi genes of S.pneumoniae, LFH-PCR was introduced to generate a gene disruption construct consisting of erm cassette with long flanking homology regions to the target gene. Then S.pneumoniae strain 31203 was transformed directly with this PCR product.The galU-deletion mutant was obtained on the TSA agar containing erythromycin and identified by PCR and sequencing. The results implicated that galU gene was completely replaced with erm cassette. Carbohydrate fermentation tests showed that galU-deletion mutant strain fermented Glu but was unable to use Gal as a carbon source. The transformation frequency of the deficient mutant at 10min induced by CSP-2 was 14.8±1.4%,there were no differences between the parent strain and the deficient mutant in efficiency of genetic transformation(P>0.05).The deficient strain was used to infect BALB/c mice to determine the virulence. After 24h,None bacteria was harvested from blood and lung tissue of infected mice, which showed the mutant strain had low survival capacity and virulence in BALB/c mice(P<0.05).So,the galU-deletion mutant strain could be used to screen in vivo-promoter in S.pneumoniae because it is incapable of synthesizing capular polysaccharide and hence loses virulence.The target gene could be completely replaced by LFH-PCR product,so this method is simple,rapid and effective in functional genome research of S.pneumoniae. 2. Construction of promoter-trap library for screening in vivo-induced genes in S. pneumoniaeTo identify in vivo-induced genes in S.pneumoniae with in vivo expression technology (IVET), a large promoter-trap library using galU and lacZ as the reporters was constructed. Based on the suicidevector pEVP3, IVET vector pEVP3-galU was constructed with promoterless galU gene as an in vivo reporter. Firstly, promoterless galU gene was directly cloned into pEVP3 and fused with promoterless lacZ gene (an in vitro reporter). Then the random pieces of S. pneumoniae chromosomal DNA (200-500bp), obtained by partial Sau3AI restriction digestion,were subcloned into the Bgl Ⅱ site of pEVP3-galU.Upon introduction of the...
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