Research On Pneumoloysin Influence On Bacterial Virulence And Raw264.7 Apoptosis

Background Pneumolysin is an important virulence factor of streptococcus penumoniae which has many biological functions such as dissolved cell activity, complement activation and apoptosis. But it remains unclear that what kind of role it will play in the process of bacterial infecting host.This research is to study in which part PLY will effect in the process of bacterial infecting host through comparing the diference of planting, intrusion and viability in blood between Ply mutant strain and wild type strain.The result shows PLY plays an important role in the process of damaging lung tissue and breaking pulmonary capillaries barrier. Recent research shows that in the lung damaging led by streptococcus pneumoniae, the effect of PLY apoptosis induction is stronger than that of cytotoxicity. While its specific molecular mechanism remains unclear. Pulmonary macrophage is an important part of this barrier and of existing research shows PLY may led macrophage apoptosis which suggested pulmonary macrophage apoptosis led by PLY is an improtant factor for lung damaging and bacteria hitting into the blood. There is research showing that this process relies on the TLR4, while its specific molecular mechanism remains unclear. The second half part of this research is to study macrophage specific molecular mechanism of apoptosis led by PLY based on mice alveolar macrophages RAW264.7 which could provide valuable data for further study for pathopoiesia molecular mechanism of Streptococcus pneumonia.Methods Adopt the way of Long flanking homology polymerase chain reaction to build Ply mutant strain, to research pneumolysin influence to bacterial virulence through mice experiment.With the RAW264.7 been processed by rarefied PLY protein, apoptosis was identified through the experiment of cell growth observation, MTT cell proliferation assay, DNA Ladder analysis and Flow cytometry. Detecting Caspase-3,8,9 activity by ELISA and analyze Bax,Fas,Bcl-2 by protein immunohistochemistry.Results Ply mutant strain was successfully built. The time of mutant strain entering the blood(6h)is obviously posterior to that of wild type strain(2h)and bacterial load of former is significantly lower than that of wild type strain at every time point, which shows out statistically significant difference (P<0.01). Mice peritoneal infection model shows that median live time of wild type strain is 3 days and that of mutant strain is 18 days, which shows out statistically significant difference(P<0.01).With RAW264.7 been processed by pneumolysin, typical change on apoptosis morphology was observed from inverted microscope. The MTT cell proliferation assay shows pneumolysin have a great cytostatic action effect on RAW264.7 with dependence on time and concentration. Analysis from FCM shows 24h and 48h's early apoptosis rate of RAW264.7 processed by 0.5 ug/ml PLY protein are 7.42% and 15.64%, compared with that processed by 1ug/ml PLY protein are 43.33% and 55.43%(P<0.05).DNA Ladder of chromosomal DNA were detected by agarose gel electrophoresis.Caspase-3,8,9 activity and related protein Bax,Fas expression rised while Bcl-2 expression reduced with RAW264.7 been processed by PLY protein..Conclusions Streptococcus pneumoniae Ply gene defect reduce bacterial's virulence and infestations in the host which suggested that PLY plays an important role in damaging lung and breaking pulmonary capillaries barrier which may refers to induction of apoptosis alveolar macrophage. The research on RAW264.7 apoptosis shows PLY may lead mice macrophage RAW264.7's apoptosis and its mechanism related to dual controls function of death receptor/Fas way and mitochondrial way.