| Elizabethkingia miricola can infect a variety of farmed and wild frogs.The meningitis-like disease caused by E.miricola in frogs is a contagious and fatal disease that causes serious economic losses in frogs industries in China.Therefore,it is essential to study the pathogenic mechanism and virulence factors of E.miricola,providing theoretical basis for develop drugs and attenuated vaccine against E.miricola infection.Outer membrane protein A function in many capacities including membrane structure and stability,signal transduction,invasion and defense.However,the role of Omp A in E.miricola remains unknown.In this study,E.miricola FL160902 was used as the wild type strain and 5 ompA deletion strains were constructed successfully via homologous recombination.Further biological characteristics and pathogenicity of 5 ompA deletion strains were studyed.The main results and conclusions are as follows:E.miricola ompA deletion strains were constructed by homologous recombination method.There are 5 ompA genes in genome of E.miricola FL160902 strain,which are ompA-0703,ompA-2506,ompA-2507,ompA-3180 and ompA-3488.Construction of 5ompA deleted mutants by homologous recombination through a recombinant suicide vector p RE112-ompA-LSR.The technical platform of E.miricola gene knockout was established,which provided technical means for the study of E.miricola genes function.The biological characteristics of E.miricola ompA deletion strains were analyzed by growth characteristics,oxidative tolerance and biofilm formation ability.The growth curves of the 5 ompA deletion strains were similar to wild strain.The results of H2O2 tolerance assay showed that the antioxidant capacity of△ompA-3488 was significantly reduced（P<0.05）,indicating that ompA-3488 gene responds to oxidative stress.The crystal violet assay showed that the biofilm formation ability of△ompA-0703（P<0.001）,△ompA-3180（P<0.001）and△ompA-3488（P<0.01）were significantly enhanced,indicating that ompA-0703,ompA-3180 and ompA-3488 genes were negatively regulated the biofilm formation of E.miricola.The pathogenicity of E.miricola ompA deletion strains were analyzed by serum survival assay,adhesion and invasion assay,animal experiments.The serum survival capacity of 5 ompA deletion strains were similar to wild strain.Adhesion and invasion assay showed that the adhesion capacities of△ompA-0703（P<0.01）,△ompA-3180（P<0.001）and△ompA-3488（P<0.001）were significantly decreased compared to wild strain,but they didn’t show obvious role in invasion capacity.Furthermore,we chose of△ompA-0703,△ompA-3180,△ompA-3488 and wild strain for experimental pathogenicity test.The blood bacterial loading of black-spotted frogs infected with the ompA deletion strains were similar to those of wild strain.The spleen bacterial loading of black-spotted frogs infected with the ompA deletion strains were much lower than those of wild strain post-infection 3 day and5 day（P<0.05,P<0.0001）.The brain bacterial loading of black-spotted frogs infected with the ompA-3488 deletion strain was much lower than those of wild strain post-infection 5day（P<0.01）.The results indicated that ompA-0703,ompA-3180 and ompA-3488 genes were participated in the adhesion ability of E.miricola and affected its pathogenicity. |
