Study On Hemolysis Mechanism Of Clostridium Perfringens α Toxin

Clostridium perfringens,formerly known as C.welchii or Bacillus perfringens,is a Gram-positive bacterium with blunt ends,grouped singly or in pairs.The pathogenic factor of this bacterium is the exotoxin.It has been found to produce more than 20 kinds of exotoxin.According to the six major exotoxins(α,β,ε,ι,CPE,and Net B),it can be classified into seven types from A to G.Among them,alpha toxin(CPA)can be produced by all types of Clostridium perfringens,mainly leading to yellow sheep disease and food poisoning and gas gangrene in human.Among them,gas gangrene caused by CPA has the characteristic of rapid onset,rapid progress,lack of specific drugs,poor prognosis and high mortality,which has a great impact on industried production and human health.Meanwhile,CPA,as a potential biotoxin warfare agent,has been listed in the Control list of dual-use biological agents in China and the international community.Therefore,promoting the study of CPA is of great biosafety value to the military and medical fields of our country and even the world.Currently,studies on CPA are limited to its role as phospholipase.It is generally believed that CPA causes hemolysis mainly because it hydrolyzes cell membrane,leading to RBC rupture and causing hemolysis.The molecular mechanism of CPA binding to RBC leading to hemolysis is still lacking.Therefore,this study aims to reveal the molecular mechanism of CPA-induced hemolysis and ultimately provides a theoretical basis for the clinical treatment of CPA-induced diseases by taking human and mouse red blood cells as research objects.First,the recombinant toxin protein r CPA was expressed by p TIG-his-cpa(C-terminal containing 6 × His tag)plasmid stored in our laboratory and purified by using His Trap Chelating HP.The purified r CPA was concentrated by ultrafiltration,and its purity and properties were validated by SDS-PAGE and Western bloting.Finally,the protein concentration was determined by BCA method and stored at 4 ℃ for later use.Then,human and murine red blood cells were used as experimental subjects to detect the hemolysis activity of r CPA,and the effects of different toxin concentrations,reaction time and reaction temperature on r CPA-induced hemolysis were explored.The results showed that r CPA and human red blood cells incubated at 37 ℃ for 48 h could lead to hemolysis of 80%,and about 90% of the murine red blood cells.In addition,temperature had an important effect on r CPA-induced hemolysis,and obvious hemolysis inhibition occurred at 25 ℃ and 4 ℃,while there was no significant difference between 37 ℃ and 42 ℃.By scanning electron microscope,we observed the morphological changes of human red blood cells after r CPA treatment.We found that after r CPA treatment,red blood cells had obviously morphology changes.These changes included volume shrinkage,appearing surface sag and protuberant,and the dissolution of red blood cells over time.Based on the above studies,we further explored the molecular mechanism of r CPA-induced hemolysis.In recent years,many studies have shown that some pore-forming toxins,such as Clostridium perfringens ε toxin,Staphylococcus aureus αtoxin and Escherichia coli α hemolysin,are closely related to P2 receptor activation.However,there is still a lack of studies on the correlation between P2 receptor and hemolysis caused by non-pore-forming toxins.When non-selective ATP receptor(P2receptor)inhibitors(PPADS)were applied to the r CPA-induced hemolysis system,it was found that the hemolysis ability of r CPA on human and murine erythrocytes could be totally inhibited by PPADS,and the inhibition ability gradually increased with the increasing concentration of PPADS.This phenomenon demonstrated that P2 receptor is closely related to r CPA-induced hemolysis.Next,we investigated the effect of multiple selective P2 receptor inhibitors on r CPA-induced hemolysis.Inhibitor results showed that in r CPA-induced hemolysis,the key role of P2 receptor subtypes in human erythrocytes are P2X7 and P2Y13;The subtypes that play key roles in murine erythrocytes are P2X1 and P2Y13.It has also been reported that ATP is involved in the hemolysis process of some pore-forming toxins.When ATP is released from the forming pores,it activates P2 receptor,which interacts with pannexin1 channel to form P2-Pannexin1 complex,increasing ATP release and further promoting hemolysis.Therefore,we investigated the role of extracellular ATP in r CPA-induced hemolysis.The results showed that a large amount of ATP was released in the early stage of r CPA-induced hemolysis and reached to the maximum at about 5min.When we degraded extracellular ATP using the ATP-degrading enzyme adenosine triphosphate bisphosphatase(Apyrase),we found that it had no effect on the hemolysis of r CPA.When pannexin1 channel protein inhibitors Carbenoxolone and Mefloquine were further used to inhibit the activation of Pannexin1 channel protein,they also had no effect on r CPA-induced hemolysis.Thus,P2 receptor activation is independent of extracellular ATP and P2-pannexin1 complex formation.To provide insights into the downstream pathways that may be affected by P2 receptor activation,we used untargeted metabolomics analysis to determine the changes in metabolites after r CPA was applied to human erythrocytes.The results showed that the main metabolic pathways affected by r CPA were alanine,aspartic acid and glutamic acid.Through literature search,we found that this pathway is closely related to the generation of intracellular reactive oxygen species,and because of no mitochondria in red blood cells,the reactive oxygen species are mainly generated by NADPH oxidase.Therefore,we used NADPH oxidase inhibitor Diphenyleneiodonium chloride(DPI),and finally confirmed the key role of NADPH oxidase in r CPA-induced hemolysis.Finally,according to the activation pathway of NADPH oxidase and the mechanism of DPI,we verified it with a variety of inhibitors,and finally preliminarily revealed the pathway that P2 receptor activation leads to reactive oxygen species generation.Previous work in our laboratory confirmed that pore-forming toxin ETX can only cause hemolysis of human erythrocytes,and ETX-induced hemolysis is closely related to P2X7 and P2Y13 receptors of human erythrocytes.Therefore,we speculate that there may be a common P2 receptor-based hemolysis pathway between pore-forming toxins and non-pore-forming toxins.To verify this hypothesis,ETX with fluorescence label was constructed,and uesd to verify the early studies on the hemolysis of ETX.By constructing Staphylococcus aureus b hemolysin(Hlb),we proved that another non-pore-forming hemolysin Hlb is also closely related to P2 receptor.In conclusion,our study for the first time found the inhibitory effect of P2 receptor inhibitors on r CPA-induced hemolysis,identified the key P2 receptor subtypes affecting r CPA-induced hemolysis,and preliminarily revealed the signaling pathway that r CPA activated P2 receptor,led to the production of reactive oxygen species,and ultimately led to hemolysis.These findings have changed the traditional perception of the hemolysis mechanism of r CPA,and proved that there may be a universal P2receptor-based hemolysis pathway between pore-forming toxins and non-pore-forming toxin-induced hemolysis,providing ideas and guarantees for the treatment of CPA-induced diseases and national biosafety.