The Research On The Relationship Between The Pore-forming Activity Of Pyolysin Of Trueperella Pyogenes And Its Ability In Inducing Apoptosis Of Cultured Macrophages

Trueperella pyogenes?T.pyogenes?is a conditional pathogen of humans and various animals.T.pyogenes is one of the important pathogens causing pneumonia,mastitis,endometritis and other organs and mucous purulent infections.Animals can be dead through septicemia when state of an illness is serious.That condition can cause severe damage to breeding industry.Pyolysin?PLO?is an exotoxin and the only hemolysin secreted by T.pyogenes.As one member of the cholesterol-dependent cytolysin?CDCs?family,the cytolytic effect can be exerted by binding to cholesterol in the cell membrane to form a transmembrane channel.However,the role of PLO in T.pyogenes infection has not been fully elucidated.The study first inoculated mice with T.pyogenes through the respiratory tract.The results showed that T.pyogenes can proliferate in mouse lungs and spread to other organs,eventually lead to animal death.Western blot analysis showed that T.pyogenes infection can induce caspase-3 and caspase-9 activation in mouse spleen and lung.While T.pyogenes and inactivated T.pyogenes were used to stimulate mouse macrophages,it was found that T.pyogenes can activate caspase-3,whereas inactivated T.pyogenes cannot.The above results indicate that T.pyogenes infection can cause apoptosis in mouse cells,and these effects may be related to PLO,an exotoxin secreted by T.pyogenes.In this study,rPLO D238R was prepared by replaced the 238th aspartic acid?D?of recombinant PLO?rPLO?with arginine?R?.The assay showed that the hemolytic activity of rPLO D238R was lower than rPLO significantly.The identification of rPLO D238R showed that its binding ability to cell membranes was similar to rPLO,but the ability to form high molecular weight oligomers on cholesterol-containing liposomes was attenuated significantly.Transmission Electron Microscope?TEM?observed that rPLO can form a typical transmembrane pore structure on cell membranes,while rPLO D238R cannot.In addition,the toxicity of rPLO D238R to L929and MDBK cells was also significantly lower than that of rPLO.The above results indicate that the pore-forming activity and cytolytic activity of rPLO are closely related to its membrane-binding ability and oligomerization ability.In this study,it was found that when rPLO was applied to mouse macrophages,it resulted in a significant increase in the activation of caspase-2/3/6/8/9 in cells;Western blot results showed that PLO-treated macrophages can cause activation of caspase-3/9/12,rPLO D238R and rPLO P499F?another rPLO mutant's ability to binding cell membranes and hemolytic activity was significantly reduced than original PLO?did not cause above phenomenon.Above results indicate that the pore-forming activity of PLO plays a key role in its induction of macrophage apoptosis.Western blot experiments showed that rPLO can affect the mitochondrial membrane potential???m?and the release of cytochrome C?Cytochrome C?in mitochondria after exposure to mouse macrophages,and it can also cause the release of reactive oxygen species?ROS?in the cells.The above results indicate that PLO-induced apoptosis of mouse macrophages is associated with activation of the apoptotic mitochondrial pathway.Z-LEHD-FMK?caspase-9 inhibitor?,Z-VAD-FMK?caspase inhibitor?,BAPTA-AM(intracellular Ca²⁺chelator),and NAC?ROS eliminator?and rPLO are co-treated with mouse macrophages.The cells were found to inhibit the rPLO-induced activation of caspase-3.The above results demonstrate that the mitochondrial pathway is involved in PLO-induced macrophage apoptosis in mice.Taken together,this study confirms that PLO secreted by T.pyogenes can activate caspase-3and apoptosis in macrophages by activating the mitochondrial pathway,and channel.this ability is related to the ability of PLO bind with cell membrane and oligomerizes at the cell membrane.It is directly related in the ability to form the transmembrane channel.The results of this study provide a partial basis for a comprehensive understanding of the mechanism of PLO virulence effects during T.pyogenes infection.