Study On Toxicity And Molecular Mechanism Of Clostridium Perfringens ε Toxin

Objectiveεtoxin（ETX）plays a key role in the pathogenesis of Clostridium perfringens type B and D,causing damage to animal husbandry in the world.However,its pathogenic mechanism has not been fully elucidated.Therefore,it would do favor to figure out the pathological process of ETX and develop novel therapeutic targets by further exploration of the mechanism of ETX-induced hemolysis of erythrocytes and the toxicity of ETX accumulation in vivo,providing a theoretical basis for the prevention and treatment of Clostridium perfringens infection withεtoxin gene.Methods1. To observe whether ETX could damage to multiple organs,we constructed the recombinant plasmid vectors of p TIG-His-ETX and p TIG-mscarlet-ETX-His and extracted 6×His-ETX and mscarlet-ETX proteins,respectively.After injected into the mice via the tail vein,the accumulation of m Scarlet-ETX in the organs of mice was observed through in vivo imaging system.After His-ETX was injected through the tail vein,the organ tissues were removed to observe whether it would bind with different organs and small intestine segments by immunohistochemistry.The pathological damage of His-ETX to different organs was observed by HE,and the binding of m Scarlet-ETX to different small intestine segments was verified by immunofluorescence.2. To investigate the molecular mechanism of human red blood cell hemolysis caused by ETX,human erythrocytes were stained with PKH26 to observe the effect of ETX on human erythrocytes volume with laser confocal scanning microscope.At the same time,changes in Ca²⁺influx,ceramide abundance,phosphatidylserine eversion,and reactive oxygen species in erythrocytes after ETX treatment were detected by flow cytometry.Then we verified the expression of MAL in human,mouse and rat red blood cells and HEL cells by immunofluorescence,Western blot and flow cytometry.The expression of MAL protein in HEL cells was blocked by Lentivirus-mediated RNA interference technology,and then to verify whether MAL protein was involved in ETX-induced cytotoxicity with PI incorporation,MTS,Western blot,and immunofluorescence techniques.Finally,GST pull-down assay was carried out to verify whether the MAL protein in human red blood cells and HEL cells could bind ETX in vitro.3.To investigate whether ETX has toxic effects on skin and related cells,we verified the toxic effects of ETX on Ha Ca T and HEKs cells through MTS,m Scarlet-ETX binding,ATP release,oligomer and pore formation assays.The expression of MAL protein in Ha Ca T and HEKs cells was blocked by Lentivirus-mediated RNA interference technology,and then MTS and immunofluorescence assays were performed to verify whether MAL protein was involved in ETX-induced cytotoxicity.We calculated the 50%lethal dose of ETX by subcutaneously injection of His-ETX of different concentrations.The spread of ETX toxin and pathological changes of skin,brain and kidney were analyzed by pathological section assays.The binding of m Scarlet-ETX and the expression of MAL protein in the skin were observed by immunofluorescence.Results1.ETX causes damage to multiple organ and mainly binds to the colon in the small intestine.Overall observation of small animal through in vivo imaging system revealed that m Scarlet-ETX mainly accumulated in the brains.Subsequently,different organs were removed and placed in small animal live imagers to observe that the intensity of fluorescent signals in the brain,kidney,lung,liver,spleen,and heart of the m Scarlet-ETX group was significantly higher than control.Immunohistochemical results showed that no positive expression was found in brain,kidney,lung,liver,spleen,and heart tissues of the control group.In the ETX treatment group,the combination of ETX was found in the renal tubules and corpuscles of the kidney tissue,the white matter portion of the brain tissue,the alveoli of the lung tissue,the hepatic lobules of the liver,the parenchyma of the spleen and the cardiac membrance of the heart.HE results showed that ETX causes damage to different organs:dense nucleus and hemorrhage in the distal tubule epithelial cells of kidney tissue;edema in brain tissue;obvious expansion around the lung bronchi in lung tissue,exudation of a large amount of serous fluid;hepatic lobular necrosis in the liver,hepatocyte degeneration was observed near the necrotic area;splenocyte degeneration in the spleen,accompanied by bleeding;and obvious heart edema and bleeding.In addition,compared with the control group,it was found that His-ETX and m Scarlet-ETX mainly bind to the colon,with a small amount of binding in the rectum,jejunum,and ileum,but no combination was found in the duodenum and cecum.2. ETX triggers shrinkage of erythrocytes via MAL receptor with subsequent phosphatidylserine exposure.The results showed that ETX cause human erythrocytes to shrink and subsequently swell to rupture.Flow cytometry showed that ETX causes Ca²⁺influx,increase of ceramide abundance,and phosphatidylserine eversion in human erythrocytes,but no generation of reactive oxygen species.Immunofluorescence and western blot results showed the expression of MAL protein was only observed in human erythrocytes and HEL cells,and co-localized with the binding of m Scarlet-ETX.Compared with normal cells,it was found that interfered with MAL expression significantly reduced the cytotoxicity,pore-forming ability and oligomer formation of ETX.It was demonstrated for the first time in vitro that ETX can directly bind to MAL protein.3. ETX induces toxic effects in skin and skin cells.The results show that ETX can bind to Ha Ca T and HEKs cells and cause significant cytotoxicity.We also found that ETX could form pores and oligomers in these cells,leading to ATP release.Immunofluorescence staining revealed that m Scarlet-ETX could bind to the cell membrane in Ha Ca T and HEKs cells,and co-localized with the expression of MAL protein in the cell membrane.Compared with normal cells,it was found that ETX did not significantly affect the cytotoxicity and pore-forming ability of cells after MAL block.The LD₅₀ of mice dying by subcutaneous injection of ETX was 109 ng/kg,with edema of the hair follicles and muscle layers.ETX does not spread to the surrounding area under the skin,whereas penetrating into the internal organs directly and causing edema and bleeding of brain and kidney.And immunofluorescence results showed that m Scarlet-ETX bind with MAL protein expression in human and mouse skin.Conclusion Firstly,it was confirmed that m Scarlet-ETX could accumulated in the brain,kidney,lung,liver,spleen,and heart tissues of mice and ETX bind with these organs causing significant pathological changes,especially ETX is mainly binding to the colon of small intestine.It has been confirmed that ETX caused damage to integral organ and the absorption of ETX is mainly in the colon.Secondly,it was found that ETX caused human erythrocytes to shrink at first,and then swelled until ruptures,followed with Ca²⁺influx,increased ceramide abundance,and phosphatidylserine eversion.The binding of m Scarlet-ETX and the expression of MAL protein was co-localization on human erythrocytes.MAL protein was involved in the cytotoxicity of HEL cells,the formation of pores and oligomers,and ETX could bind to MAL protein in human erythrocytes and HEL cells in vitro.It was speculated that MAL protein played an important role in ETX-induced hemolysis;Finally,it was found that ETX could cause the death of Ha Ca T and HEKs cells.The subcutaneous injection of ETX at LD50 was 109 ng/kg and caused edema of the hair follicles and muscle layers in the skin.The binding of ETX on skin tissue may be related to the expression of MAL protein,but MAL protein does not involve in the toxic effect of ETX on Ha Ca T and HEKs cells.These data provide a theoretical basis for the prevention and treatment of Clostridium perfringens infection throughεtoxin gene.