Screening Of Pneumococcal Virulence Genes Regulated By Transformation

Among the gram-positive opportunistic human pathogen, Streptococcus pneumoniae(S.pn) 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 in immunocompromised patients. S.pn 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.Natural transformation is a kind of gene transfer manners in bacteria. According to some papers, pneumococcal transformation was probably related to its virulence and against.In the present work, we employed the differential fluorescence induction and identified the genes expressed in the process of pathogenesis due to S.pn, which were regulated by natural transformation. These genes might be related with bacterial opportunistic infection. The research work included the following three parts. 1. Construction and analysis of promoter-trap library of S.pn. The promoter-trap library is very important to screen the in vivo-induced genes by DFI. 200 to 800bp fragments of S.pn TIGR4 DNA were cloned upstream of the promoterless gfp gene in pEVP3-SDGFP. The recombinant plasmid was then transformed into E.coli DH5α.47 000 recombinants were obtained. Considering insert DNA orientation and insert size, this represents 5 coverage of the 2.2Mb S.pn genome, 90% of these clones had 200 to 800bp inserts and the library is random. Transformation by this plasmid library yielded 500 000 S.pn transformants. Analyzed 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 vivo of S.pn.2. Screening of genes induced in vivo by DFI and bioinformatics analysis. The promoter-trap library in S.pn was grown to logarithmic phase and used to infect BALB/c mice intraperitoneally. After 24 hours, bacteria were harvested from blood and lung tissue, then sorted by flow cytometry, clones with increased fluorescence were used to reinfect animals for enrichment. 64 clones were collected and analyzed. To clone the induced fragments, each chromosomal DNA was digested with BamHⅠ, then self ligated, and transformed into E.coli DH5α. All the recombined suicide plasmids were successfully obtained and sequenced, and 10 unique sequences were identified. Bioinformatics analysis showed that there were 18 ORFs in the 10 operons. These genes involve in the process of energy transport and metabolism, cation acquisition, cell envelope biogenesis, transcription, surface protein, and the functions are unknown. It is necessary for further research about whether these genes regulated by transformation of pneumococcus.3. Construction and analysis of transformation-deficient S.pn. 10 different strains of S.pn, which had been identified at the second step study, were included to construct transformation-deficient pneumococci, then were grown to logarithmic phase respectively and used to infect BALB/c mice intraperitoneally. After 24 hours, bacteria were harvested from blood and lung tissue, analyzed by flow cytometry. At last, 3 gene fragments, which involve in the process of energy metabolism,cation acquisition and the function are unknown in mice blood, were found to be regulated by natural transformation. Further investigation of these genes might lead to understand molecular pathogenic mechanism of S.pn, which provide new theory and experimental data for more effective drug and vaccine development.