| Aeromonas veronii is a Gram-negative facultative anaerobe widely distributed in aquatic environments and can cause various diseases in humans and aquatic animals.In recent years,an increasing number of cases reported both domestically and abroad have shown that the virulence of this bacterium is on the rise,posing a serious threat to China’s aquaculture industry and public health.The virulence of Aeromonas is multifactorial and multifunctional,related not only to its structural components（such as outer membrane,flagella,etc.）,but also to its secreted substances（such as hemolysin,lipase,protease,biofilm,etc.）.Its pathogenicity is the result of the synergistic action of multiple components.Traditional disease research is gradually unable to meet the needs of disease prevention and control,and it is necessary to explore the main virulence factors and their mechanisms of action of A.veronii through molecular biology and other means.In this study,we screened the differential virulence genes of two strains of A.veronii GL2（high virulence）and FO1（low virulence）isolated and preserved from diseased triangle sail clams with significant differences in virulence.It was determined that the tol C gene was significantly differentially expressed between the two strains.This study further constructed mutant strains of the tol C gene and carried out experiments on biological characteristics,related pathways of virulence genes,immune response of H.cumingii,etc.,aiming to comprehensively elucidate the function and mechanism of action of the differential virulence gene tol C.The main methods used and results obtained in this study are as follows（1）We previously isolated and preserved two strains of A.veronii,GL2 and FO1,from diseased clams with significant differences in virulence.Using techniques such as semi-quantitative PCR and real-time quantitative PCR,we analyzed the differences in the expression of virulence-related genes at the transcriptional level between these two strains.Our research found that the expression level of the gene tol C,which is related to bacterial multidrug resistance and encodes an outer membrane channel protein,was significantly higher in the high-virulence strain GL2 than in the low-virulence strain FO1.（2）We constructed a tol C gene mutant strain of A.veronii GL2 by homologous recombination.The tol C＿UD fragment was connected by overlap PCR to connect the upstream and downstream homologous arms of the tol C gene and inserted into the suicidal plasmid p RE112.E.coli WM3064 was used as a donor to transfer the recombinant suicidal plasmid p RE11-tol C＿UD to the wild-type strain GL2.The tol C gene was successfully knocked out using the principle of homologous recombination.The mutant strain named A.veroniiΔtol C was successfully constructed and verified by resistance plates and corresponding primers.Its genetic stability was confirmed,indicating that the suicide plasmid p RE112 could be used in knocking out the genes of A.veronii efficiently.（3）To explore the effect of tol C gene deletion on the biological characteristics of A.veronii GL2,we detected the growth situation,biofilm formation,drug resistance and expression of multidrug efflux pump pathway related genes between mutant strainΔtol C and wild-type strain GL2 to preliminarily understand the function of tol C gene in A.veronii.The results showed that tol C gene had no significant effect on colony morphology,growth status,hemolysis,or protease activity of A.veronii.However,deletion of tol C gene significantly increased sensitivity to cefazolin,ceftriaxone,neomycin and erythromycin,reduced multidrug resistance and biofilm formation ability,and reduced expression of acr A,acr B and mar A genes.（4）To explore the effect of tol C gene deletion on the virulence of A.veronii,we obtained the LD50 values of wild-type strain GL2（1.979×106 CFU/g）,wild-type strain FO1（5.809×107 CFU/g）and mutant strainΔtol C（2.155×107 CFU/g）for H.cumingii by semi-lethal test.The LD50 value of mutant strainΔtol C was significantly lower than that of wild-type strain.After infection with wild-type strain GL2 and mutant strainΔtol C,we found that wild-type strain GL2 could cause severe immune stress in H.cumingii and the immune enzyme activity of host reached its peak within 6 h;while mutant strainΔtol C had lower virulence and induced immune stress than GL2,and the immune enzyme activity of host after stimulation was significantly lower than that of GL2 group.Some tissues even recovered to normal level at 48 h.Wild-type strain GL2 caused serious damage to host’s hepatopancreas and gill tissues;while mutant strainΔtol C caused mild damage.The relative m RNA expression levels of Hc IL-17,Gal R2,s HSPs and TR in the test group were significantly increased after infection for 12 h.In summary,this study aimed at the phenomenon that there were significant differences in virulence and gene expression between A.veronii GL2 and FO1,and verified that the outer membrane channel protein Tol C was one of the important factors regulating the virulence of A.veronii by screening and constructing gene deletion strains.Its mechanism of action may be related to the secretion of virulence-related substances regulated by Acr AB-Tol C multidrug resistance efflux pump.This study revealed that the outer membrane channel protein Tol C played a key role in the infection process of A.veronii to the host.The deletion of tol C gene significantly reduced the virulence,biofilm formation ability and multidrug resistance of A.veronii GL2,thus reducing the immune damage caused by GL2 to the host.This provides a basis for further elucidating the pathogenic mechanism of A.veronii. |
