Construction And Characterization Of Type Ⅳ Pili Deleted Mutant Strain In Aeromonas Schubertii

Aeromonas schubertiiis associated with multi-organ necrosis disease that frequently infects cultured snakehead fishin recent years.The disease has rapid onset,short course of disease and high mortality rate,which bring great harm to the Channidae fish breeding industry.At present,the researches about A.schubertii are mainly focused on isolation and identification of the bacteria,drug sensitivity analysis,there are rarely reports about the virulence factors and pathogenic mechanisms of A.schubertii.TypeⅣpili（T4P）is an important virulence factor,which plays an important role in the pathogenic process of many pathogenic bacteria.In this study,T4P was found in Aeromonas schubertii.The gene cluster of pilM/N/O/P/Q was knocked out using homologous recombination gene editing technology to construct T4P mutant strainΔpili,and the biological characteristics and functions in the pathogenesis process ofΔpili were studied.The results were as follows:We observed bundle-forming pili that protruded from the polar surfaces of wild-type strain WL-2 by transmission electron microscopy.Moreover,we subjected A.schubertii to a pili purification procedure and the pili-specific band at 22 k Da in the wild-type strain.Combining the results of previous researches,it was confirmed that the bundle-forming pili were T4P.The mutant strainΔpili was constructed by homologous recombination gene editing technology.The deletion result of the pilM/N/O/P/Q gene cluster was confirmed by PCR and sequencing.The pilM/N/O/P/Q gene cluster was not expressed fromΔpili by Real-time PCR.The bundle-forming pili was observed on surface of the mutantΔpili by transmission electron microscopy.We subjectedΔpili strain to a pili purification procedure and the pili-specific band at 22 k Da was clearly absent in theΔpili strain.In addition,the study also found that theΔpili strain had no significant difference in colony morphology,hemolysis,growth curve with wild-type strain.This study found that theΔpili strain is more sensitive to acid environments and high-salt environments compared to the WL-2 strain.TheΔpili strain was more resistant to 100 m M H₂O₂than the wild-type strain as indicated by greater survival under this stress.The ability of biofilm formation was enhanced in theΔpili strain.In addition,theΔpili strain showed higher resistance to macrophages than the wild-type strain.In this study,it was found that the twitching motility ability and cell adhesion ability were significant weakened in theΔpili strain.It was also found that delivery of theΔpili strain by immersion resulted in reduced cumulative morbidity,whereas delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild-type strain.The effects of T4P on host immune responses were assessed.It was found that expression of six immune-related genes were roughly similar between theΔpili and wild-type strain treatment groups.The expression of IL-1 m RNA in both spleen and kidney was the overall highest among the six genes following infection.The expression of IFN-γm RNA was up-regulated slowly.The expression of TCR and CD4 genes were showed a steady trend until the end of sampling,and the expression of CC22 and CD40 genes were showed a trend of rapid up-regulation and slow down-regulation at the beginning of infection.In summary,for the first time,type Ⅳ pili were discovered in A.schubertii.The homologous recombination technique was used to knock out the T4P gene cluster pilM/N/O/P/Q.The mutant strainΔpili,which the T4P gene cluster pilM/N/O/P/Q was knocked out by the homologous recombination technique,was successfully constructed.Differences in biological characteristics,pathogenic characteristics of fish,and non-specific immune response were compared between wild-type strain andΔpili strain.The results of this study provide an important reference for the research on the pathogenic mechanism of A.schubertii and the development of disease prevention and control products.