Construction of ply gene-deletion mutant of Streptococcus pneumoniae and research of its virulence change

Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced completely by erm gene. The ply deficient strain was successfully constructed. The growth of single strain culture medium showed that ply genetic defect made no influence on bacterial's external growth. While in the mice nasal cavity infecting experiment, deficient strain enter into blood after 6 h from infecting which obviously slower than that did wild-type(2 h). And the number of bacteria at each point was much smaller than that of wild-type(P ＜0. 01 ). The mice peritonaeum infecting experiment showed that median lethal time of wild-type was 3 d, while that of deficient strain was 18 d(P＜0. 01). Conclusion It is a good way to completely substitute ply gene using LFH-PCR. ply deletion made no influence on baterial's growth in vitro, but it resulting in reduction of bacterial virulence in vivo. Key words: Streptococcus pneumoniae;  Pneumolysin;  LFH-PCR;  Virulence