The Mechanism Of Pyroptosis Induced By Clostridium Perfringens ?1 Toxin In Macrophages

Clostridium perfringens is the most widely distributed pathogen in nature,as an opportunistic pathogen,it can act causing diseases such as food poisoning and gas gangrene,necrotizing enteritis and enterotoxemia in humans and domestic animals.The bacteria produce several toxin proteins which play key roles in the pathogenesis of Clostridium perfringens infection.The ?1 toxin is one of the lethal toxins of Clostridium perfringens.Clostridium perfringens ?1 toxin is responsible for necrotizing enteritis and enterotoxemia.Pyroptosis is an inflammatory form of programmed cell death.However,it is unclear whether Clostridium perfringens ?1 toxin regulates host cell damage.In order to better to detect,prevent and treatment of diseases caused by Clostridium perfringens infections,it is a necessity to investigate the cytotoxicity and pathogenesis of Clostridium perfringens ?1 toxin.In this study,the obtain of recombinant Clostridium perfringens ?1 toxin(rCPB1)by using gene recombination technology,and cytotoxic activity of purified rCPB 1 toxin was assessed by CKK-8 assay.By analyzing the expression changes of pyroptosis related signal molecules in each cell after the action of rCPB1 on macrophages.The Caspase 1 interference model of RAW264.7 cells was established to explore the pyroptosis pathway of macrophages caused by ?1 toxin.A comprehensive analysis of mechanisms of biological toxicity of CPB1 and the cell injury mechanism,to provide a theoretical basis for further revealing the possible pathogenic mechanism of animal enteritis caused by Clostridium perfringens infection.Following results have been achieved after an accomplishment of this study:(1)Clostridium perfringens ?1 toxin was successfully generated by using gene recombination technology,after the optimization of induction and denatured the inclusion body.The cytotoxic activity of rCPB1 toxin was assessed using RAW264.7,THP-1 macrophages in terms of CKK-8 assay,and the ID50 was about 25 ?g/mL,22 pg/mL.These results showed that rCPB1 toxin was successfully generated.(2)The results showed that the expression leves of NLRP3,Caspase 1,Gasdermin D,IL-18 and IL-1? were up-regulated after RAW264.7,THP-1 macrophages and BMDM(Bone Marrow-derived Macrophages)were treated by rCPB1.Indicating that rCPB1 could induce pyroptosis in macrophages cells.(3)The Caspase1 interference model of RAW264.7 cells was established.After the use of small interfering RNA to interfere with the expression of Caspase1,the rupture of the plasma membrane of RAW264.7 treated by rCPB1 could be alleviated,and the formation of intracellular cavitoids could be reduced.Moreover,the expression of Caspasel,Gasdermin D,IL-18 and IL-1? in RAW264.7 co-treated by rCPB1 and siRNA caspase1 could be down-regulated compared to the groups treated by rCPBl.It suggests that rCPB1 may induce pyroptosis in RAW264.7 cells through the caspase1-dependent classical pyroptosis pathway.In conclusion,we prove that after rCPBl treated on macrophages,it promotes the assembed of NLRP3 inflammasomes,activates Caspase 1,and the activated Caspase 1 cuted Gasdermin D to form plasma membrane pores,leading to the release of inflammatory factors IL-18 and IL-1?,resulting in macrophage pyroptosis.The results of this study will provide a new research direction for the pathogenesis of clostridium perfringens ?1 toxin.