The Cytotoxicity And Pathogenesis Of Beta2 Toxin Of Clostridium Perfringens

Clostridium perfringens is an important agent of both enteric and cytotoxic infections in humans and domestic animals.The bacteria produce several toxin proteins which play key roles in the pathogenesis of Clostridium perfringens infection.The Beta2 toxin as major lethal exotoxins of Clostridium perfringens(CPB2),however,the cytotoxicity and pathogenesis of this exotoxin remain largely unknown.In order to better to detection,prevention,control and treatment of diseases caused by Clostridium perfringens infections,it is a necessity to investigate the cytotoxicity and pathogenesis of CPB2.To this end,the ultimate goal of this study aimed to prepare purified recombinant CPB2(rCPB2)using gene recombination technology,sequentially generate monoclonal antibodies to CPB2 and trace the action of CPB2 on its target cells using antibody;Meanwhile access the cell apoptosis,production of ROS and NO,mitochondrial damage,expression of apoptotic signaling molecules,such as BCL-2 and Caspase protein families in target cells treated with rCPB2,in order to determine the cytotoxicity of CPB2 and uncover the molecular mechanism of cell injury induced by this toxin;finally analyze cytokines secreted by Thl/Th2/Th17 cell subpopulation in the sera,intestinal tissues and spleens of mice administrated with rCPB2 via tail vein or intragastric delivery.Together with aforementioned cytotoxicity of CPB2 and its potential mechanism of cell injury,a comprehensive analysis of mechanisms of biological toxicity of CPB2 in vitro and in vivo will allow us reveal the cell injury mechanism of CPB2,demonstrate the potential pathogenic mechanism of enteritis in animals caused by Clostridium perfringens infections,which will provide an insight for further investigation in the pathogenesis of Clostridium perfringens infection and immunological prevention for this disease.Following results have been achieved after an accomplishment of this study:1.In the present study,a recombinant His-tagged C.perfrigens beta2(rCPB2)toxin was successfully generated and characterized.The rCPB2 was further purified using AKTA purification system,owing to its ability to bind to Ni-NTA column.The resultant purified rCPB2 showed the integrity with cytotoxicity in vitro.The cytotoxic activity of purified rCPB2 toxin was assessed using NCM460 cells in terms of MTT assay.The cytotoxic activity was observed at a concentration as low as 5.0 ?g/ml,and the concentration of rCPB2 causing 50%cell death(ID50)was determined by 15 ?/ml.2.Since studies on the pathogenic roles and functional mechanisms have largely been hampered by the difficulty in purification of native toxins and lack of specific antibodies to CPB2.Three hybridoma cell lines producing monoclonal antibodies(McAbs),namely 1E23,2G7 and 2H7 were generated and characterized.They were able to crossreact with both native CPB2 and rCPB2 by assays including immunoblotting,ELISA,immunofluorescent staining and neutralization analysis.Importantly,these antibodies were capable of neutralizing the rCPB2 cytotoxicity on NCM460 cells.Mechanistically,rCPB2 was able to first bind to the cell membrane and dynamically translocate into the cytoplasm for its cytotoxic activity.These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future,which warrant for further investigations.3.The rCPB2 could induce apoptotic cell death in NCM460 cells,and flow cytometric analysis showed that the majority of cell death induced by rCPB2 was cell apoptosis rather than a necrotic death on this type of cells.The apoptotic NCM460 cell death induced by rCPB2 was in a dose-and time-dependent manner,which was also in a Caspase-dependent manner.The molecular analysis further revealed that the rCPB2-induced apoptosis was accompanied by an up-regulation of pro-apoptotic proteins Bax,PUMA and cleaved Caspase-3,Caspase7 and Caspase 9,and down-regulation of anti-apoptotic proteins Bcl-xl,Mcl-1 and phospho-Bcl-2.4.An administration of rCPB2 in mice via a route of tail vein injection or intragastric delivery led an increased concentration of cytokines including IL-1?,IL-2,IL-3,IL-5,IL-6,IL-10,IL-17,IL-23,GM-CSF and TNF-a in sera,intestine tissues and spleens in comparison with untreated mice,as determined by cytokine liquid Chip system(P<0.05).In conclusion,findings from this study provide important information on mechanisms of biological functions of CPB2 for the first time,an indicative of usefulness of the rCPB2 toxin protein and antibodies.Comprehensive evaluation and analysis of results about the biological toxicity of CPB2 in cell models and animal models will help us explore its potential mechanism in cell injury and pathogenesis of enteritis in animals.Which lays a foundation for further study in the pathogenesis of enterotoxemic diseases causes by Clostridium perfringens infection.