F199E Substitution Reduced Toxicity Of Clostridium Perfringens Epsilon Toxin By Depriving The Receptor Binding Capability

Clostridium perfringens,widely distributed on the earth and in the intestines of some animals,is an important zoonotic pathogen.Clostridium perfringens is able to produce at least fifteen exotoxins.Clostridium perfringens is divided into five types,namely,A?B?C?D and E according to the main four lethal toxins?????and?.Epsilon toxin?ETX?,produced by types B and D strains of Clostridium perfringens,is a strong pathogenic factor.ETX is responsible for fatal enterotoxemia in sheep,goat and other sensitive animals.Enterotoxemia could cause the death of animals in a short time and antibiotic treatment are unlikely to be effective,which causes serious economic losses to the farming industry worldwide.Therefore,it is essential to do the research on ETX.Our collegues previously developed a mutant protein rETX^F199E,which is of low-toxicity but retains its immunogenicity.The mutant rETX^F199E could protect the immunized mice against a challenge of a 100×LD₅₀ dose of recombinant wild-type ETX.However,the specific mechanism of the attenuated rETX^F199E remains unknown.To investigate whether the 199th amino acid residue is involved in maintaining ETX biological activity,we performed a series of experiments including testing the cytotoxicity of recombinant toxins toward MDCK cells,binding of toxins to MDCK cells,assessing the ability of rETX to form pores,and analysis of the structure change of the mutant.Firstly,the recombinant vectors pTIG-ETX and pTIG-ETX^F199E?N-terminal with 6×His?were obtained using the recombinant plasmid pTIG-ETX and pTIG-ETX^F199E?C-terminal with6×His?as the template in our lab.The four recombinant proteins were expressed in E.coli BL21?DE3?,and purified by affinity chromatography.Both rETX^F199E and rETX exhibited a high-level expression in soluble form.However,the level of soluble expression of rETX and rETX^F199E with His-tag at the C-terminal was higher than that with a His-tag at the N-terminal.The recombinant proteins with His-tag at the C-terminal or N-terminal have similar biological activity?p>0.05?.The cytotoxicity of purified proteins was measured using MTS and the value of CT₅₀ was calculated using the improved Karber's method.The result showed that the CT₅₀of rETX^F199E was 99,581 ng/mL and the CT₅₀ of rETX was 180 ng/mL.The cytotoxicity of rETX^F199E on MDCK cells was approximately 553-fold less than that of rETX.Secondly,the thermal stability and circular dichroism?CD?were performed to analyze the structural difference between rETX and rETX^F199E.The melting temperature?T_m?and fluorescence signal were used to analyze the structural changes of mutant protein.According to the manufacturer's instructions,diluted toxins were mixed with thermal shift dye.The fluorescence signal was determined using the StepOnePlus Real-Time PCR system?Applied Biosystem,UnitedStates?with a thermal gradient from 25°C to 99°C.The result showed that the T_m of rETX^F199E199E and rETX are 50.00±0.88°C and 60.95±0.33°C,respectively.The melting temperature of rETX^F199E was determined to be significantly different from that of wild-type epsilon toxin,indicating that some alterations of the structures of rETX^F199E had probably occurred,when compared with the wild-type ETX.To figure out whether the amino acid residue F199 mutation affects the secondary structure of ETX,circular dichroism spectroscopy was performed.Diluted protein was added to 1mm path length cuvette and CD spectra was obtained in the far UV region?185–260 nm?.The CD spectra of the two proteins showed a similar degree of ultraviolet absorption between 200 nm and 260 nm.Compared with the wild-type toxin,the spectrum of mutant protein rETX^F199E has a small deviation between 185 nm and 200 nm.To further evaluate the secondary structures proportion of the two proteins,the spectras were analyzed using the CDNN software.The wild-type and mutant proteins were predicted to have similar compositions of?-helix,?-strand,?-turns,and unordered structure?p<0.05?.No obvious differences were observed between the secondary structures proportion of the two proteins.This result indicated that the amino acid residue F199E substitution had no influence on the secondary structures of ETX.Thirdly,it was reported that the MDCK cells were sensitive to ETX,therefore,MDCK cell line was used in binding assays of the recombinant toxins.MDCK cells were mixed with toxins,which was fixed with 4%paraformaldehyde and blocked with BSA?5%?.Mouse anti-His monoclonal antibody and FITC conjugated goat anti-mouse IgG were utilized to detect the bound protein.The fluorescence signal was detected using the Varioskan Flash Multiplate Reader?Thermo Scientific,United States?and IX81 confocal microscopy?Olympus,Japan?to figure out the binding ability of rETX^F199E to MDCK cells.The result showed that the binding ability of rETX^F199E to MDCK cells was substantially reduced than that of rETX?p<0.05?.The binding ability of rETX^F199E was almost abolished.It showed that amino acid residue F199E substitution had effects on the binding ability of ETX to MDCK cells.The confocal microscopy assay was performed and the results are in agreement with that of the On-Cell Western assay.Next,a western blot assay was performed to test whether the amino acid residue F199E substitution could prevent the toxin from heptamer formation.MDCK cells were treated with toxins,and then the cells were solubilized and analyzed by immunoblotting.The result showed that wild-type epsilon toxin could bind to cells and formed heptamers,approximately 200 kDa.However,rETX^F199E199E was defective in heptamer formation.It probably suggested that the amino acid residue F199E substitution affected the binding ability of ETX to MDCK cells and the heptamer formation of epsilon toxin.ETX,a pore-forming toxin,could form heptamer formation in the plasma membrane and lead to an increases in intracellular Ca²⁺.Pore-forming assay was performed.Toxins were diluted with HBSS containing calcium chloride and added to MDCK cells containing Fluo-8^?AM.Fluorescence signals associated with Ca²⁺concentration were determined using Varioskan Flash Multiplate Reader.It showed that rETX induced a significant increase in the intracellular Ca²⁺concentration in MDCK cells when the extracellular Ca²⁺was available?p<0.05?.However,the rETX^F199E mutant protein could not lead to an increase in intracellular Ca²⁺concentration.It suggested that the amino acid residue F199E substitution alleviated ETX toxicity,mainly by inhibiting the binding ability of ETX and thereby inhibiting the ability of heptamer-forming process.ETX was still able to induce a significant increase in the intracellular Ca²⁺concentration in MDCK cells when the extracellular Ca²⁺was absent,it could be attributed to intracellular Ca²⁺stores.This result indicated that intracellular Ca²⁺stores were also involved in ETX induced cytotoxicity.Lastly,in order to further survey morphological changes in the plasma membrane of MDCK cells,scanning electron microscopy was performed.Cells were incubated with toxins,then fixed with 2.5%glutaraldehyde,dehydrated in graded alcohol and acetone,baked and plated with gold.Sags and crests were observed on the surface of MDCK cells when MDCK cells were treated with wild-type ETX,which was rarely seen on the surface of MDCK cells treated with rETX^F199E.In addition,this differences also illustrated that the amino acid residue F199E substitution has effects on ETX toxicity.In this study,the data demonstrated that F199E substitution reduced toxicity by abolishing the receptor binding capability of target cells.Howevere,the actual receptor of ETX has not been determined yet,although many studies are attempting to find the answer.Consequently,many questions remain unanswered such as the exact region of ETX that interacts with the receptor.With the inefficiency of receptor binding of rETX^F199E,this mutant protein could also be considered as a platform for receptor binding studies.Receptor studies will answer many of the questions and provide targets to develop antidotes against ETX intoxication or enterotoxemia,which should be further investigated in the future.In order to investigate the receptor of ETX,we should separate the proteins from the surface of MDCK cells,which could be combined with ETX.Co-immunoprecipitation and affinity chromatography were used to isolate putative receptor from MDCK cells,and LC-MS/MS technique was used to identify the putative proteins.Cells were incubated with rETX and ETX^F199E,and then lysed in 1%Triton X-100.Lysates were applied onto an affinity column and the binding proteins were washed by elution buffer.The diffenences of protein profiles between the two groups were analyzed using the iTRAQ?isobaric tags for relative and absolute quantitation?technique.The result showed that the four proteins CKAP₄,ERGIC-53,A₂MRAP,and Annexin A₂ are likely to be the receptor candidates of ETX.Further in vivo and in vitro experiments should be performed to clarify the interactions between ETX and the receptor candidates in the future.In summary,the mechanism of attenuated toxicity of rETX^F199E was clarified.The data demonstrated that F199E substitution reduced toxicity by abolishing the receptor binding capability of ETX on target cells.It suggested that the amino acid residue F199E substitution alleviated ETX toxicity,mainly by inhibiting the binding ability of ETX and thereby inhibiting the ability of heptamer-formation.Furthermore,we found that ETX could cause the Ca²⁺release from intracellular Ca²⁺stores,which may be an alternate pathway leading to cell death.The data also demonstrated the safety of rETX^F199E,which contributes to our previous work that rETX^F199E is potentially a good vaccine candidate for animals and humans.These findings potentially contribute to understanding the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX,using mutant proteins.