Construction And Characterization Of Cat Gene Deleted Mutant Of Aeromonas Schubertii

In recent years,sarcoidosis caused by Aeromonas schubertii（A.schubertii）broke out frequently in the cultivated s Channidae,which has become one of the main diseases in the Channidae industry with a short course of disease and a high mortality rate,causing huge economic losses.At present,there are few studies on A.schubertii,most of which focus on isolation,identification and drug sensitivity analysis.There are few reports on the virulence factors and pathogenesis of A.schubertii.Hydrogen Peroxidase is a kind of oxidase and exists widely in microorganisms,plants and animals and is also an important virulence factor of some pathogenic bacteria.Some catalase can help pathogenic bacteria resist the toxicity of peroxides in host immune cells and play a role in the process of pathogenic bacteria escaping the host’s natural defense.To study the effect of A.schubertii catalase on bacterial virulence,the homologous recombinant gene editing technology was used to construct the mutant strainΔcat,and its recombinant plasmid was constructed to prepare the complementary strain C-cat in this study.Comparing the mutant strainΔcat,the complementary strain C-cat and A.schubertii WL-2 of colony morphology、hemolysis activity、growth curve、acid base resistance ability and other biological characteristics and the catalase enzyme activity、hydrogen peroxide enzyme activity、tolerance in macrophage survival and the pathogenicity of hybrid snakehead.To explore the role of catalase in the pathogenic process of A.schubertii,and lay a foundation for the prevention and control of A.schubertii.The main research results are as follows:（1）The mutant strainΔcat and the complementary strain C-cat was successfully constructed.The cat gene of A.schubertii and chloramphenicol gene were linked into the plasmid pset4S and the thermosensitive suicide plasmid pset4S-cat was constructed,the 581bp cat gene fragment in A.schubertii WL-2 was successfully knockout and the mutant strainΔcat was constructed by homologous recombination;the recombinant cat gene plasmid p Smart-cat was transferred into the mutant strainΔcat to construct the complementary strain C-cat.In order to verify the expression of catalase in the mutant strainΔcat and the complementary strain C-cat,for genomic level,fluorescence quantitative PCR method was used to detect the expression level of cat gene with the three strains.There was no expression of cat gene in the deletion strain.The cat gene expression level of the mutant was 0.69 times than the wild strain,which confirmed the successful construction of the mutant and the complement strain.For expressional level,the CAT protein of A.schubertii was expressed by prokaryotic expression in this study,and the anti-CAT polyclonal antibody was prepared to establish Western blot method for detecting CAT protein.The established method was used to detect the three strains,and it was found that the mutant strainΔcat had no CAT protein expression,while the expression of CAT protein of WL-2 and the complement strain C-cat was 56k Da,the expression of CAT protein in the wild strain WL-2 was higher than that in the complement strain C-cat,indicating that the mutant strainΔcat and the complementary strain C-cat were successfully constructed in this study.（2）Comparative study on the biological characteristics of the mutant strainΔcat、complementary strain C-cat and A.schubertii WL-2.After cultured at 28℃for 24 h in 5%sheep blood agar plate,three strains were found to have the same colony size and the strains were gray、moist、smooth and 0.6-2.0 mm in diameter;the three strains showed weakβ-hemolysis after 48h culture;the growth characteristics of the three strains were analyzed and found that there was no significant difference in the growth curves of the three strains,and the three strains entered the logarithmic phase and the plateau phase at 2h and 16h,respectively.Comparison of biological characteristics showed that cat gene deletion had no significant correlation with the colony morphology,hemolytic characteristics and growth status between the mutant strainΔcat and A.schubertii WL-2.（3）Comparative study on pathogenicity between the mutant strainΔcat,complementary strain C-cat and A.schubertii WL-2 showed that the degradation ability of hydrogen peroxide of theΔcat was significantly lower than the WL-2 and the C-cat;the hydrogen peroxide survival test showed that the ability of resistant to 100 mmol hydrogen peroxide（H₂O₂）ofΔcat was significantly lower than WL-2 and C-cat,while the C-cat have a tolerance to H₂O₂ increased but did not return to the level of WL-2;the catalase activity test results showed that the catalase activity ofΔcat reduced,but the catalase activity of C-cat increased did not return to the level of the WL-2;The macrophage survival test of the three strains showed that the viability ofΔcat in macrophages was significantly lower than that of the wild strain WL-2（P<0.05）,and the viability of C-cat in macrophages was restored but did not reach the level of the wild strain too;the results of pathogenicity experiment on hybrid snakehead showed that the death rate of wild strain WL-2 group was 100%,the death rate of the mutant strainΔcat group was 100%,and the death rate of complementary strain C-cat group was 50%after deal with the three strains which were 1×10⁶CFU/ml at 30℃respectively for 2h.The results showed that the deletion of cat gene reduced the pathogenicity of A.schubertii,and the pathogenicity of the complement strain was restored after the supplementation,but did not reach to the level of wild strain.In conclusion,this study successfully constructed the mutant strainΔcat of A.schubertii and its complement strain C-cat.Further studies showed that the deletion of cat gene had little effect on the colony morphology,hemolytic characteristics and growth status of A.schubertii.The deletion of cat gene reduces A.schubertii’s ability to degrade hydrogen peroxide,survive in host macrophages and pathogenecity.It is speculated that the pathogenic mechanism of catalase in the process of A.schubertii infection is after being engulfed by macrophages,A.schubertii can escape the killing effect of macrophages by degrading the H₂O₂ secreted in the lysosome through its own catalase.This study provides a basis for further research on the pathogenic mechanism of A.schubertii and plays an important role in the prevention and control of A.schubertii.