Mutagenesis Of Genes PmbA And TldD In Aeromonas Veronii And Preliminary Exploration Of Their Virulence Mechanisms

Pathogenic bacteria can induce bacterial plague and even cause significant harm to the cultivation of Hyriopsis cumingii.With the deepening of research,powerful sequencing techniques can be applied to predict the function of virulence factors in pathogenic bacteria,but their specific mechanisms are unclear currently.In addition,previous research only paid attentions to the virulence of pathogenic bacteria and host immunity,but it cannot meet the need of disease control.Therefore,it is necessary to explore the main virulence factors of pathogenic bacteria and their mechanisms through molecular biology and other means,bringing about new ideas for the development of live attenuated vaccines in future.In order to clarify the virulence mechanisms of metalloprotease genes pmbA and tldD in A.veronii GL2from H.cumingii comprehensively,we conducted following experiments including the construction of mutants,biological characteristics,immune response of H.cumingii,and metalloprotease-related pathways.Methods in this study and the relevant results are as follows:1)Homologous recombination was used to construct mutant strains.Targetting at pmbA and tldD genes in the genome of GL2,primers pmbA-F1/R1,pmbA-F2/R2,and tldD-F1/R1,tldD-F2/R2 were designed respectively,and the fragments of upstream and downstream homologous arm were amplified.The ligation of the fragments was conducted through overlap PCR,and the products（pmbA＿UD,tldD＿UD）were inserted into suicide plasmid p RE112.Then the recombinant plasmids were transformed into Escherichia coli WM3064,and the donors WM3064 were mated into the recipient strains GL2 by ori T site on the plasmid respectively.Positive transformants were screened out on plates containing ampicillin（Amp,50μg/m L）and chloramphenicol（Cm,28μg/m L）,then colony PCR was used to verify their authenticities.Subsequently,sucrose lethal gene（sac B）on the plasmid was considered as a screening marker,and suspected mutants were screened on plates containing 10%sucrose and 50μg/m L Amp.Bacterial liquid PCR was performed using primers pmbA-F1/R2,pmbA-INF-F/R,tldD-F1/R2,and tldD-INF-F/R.Finally,single mutantsΔPmb A、ΔTld D and double mutantΔP&T were successfully screened out,and the genetic stabilities of the mutants were confirmed,indicating that the suicide plasmid p RE112 could be used in knocking out the genes of A.veronii efficiently.2)To evaluate the adverse effects of gene deletions on GL2 briefly,biological characterizations were observed in this study.The results of colony morphology observation and growth curve showed that there existed no differences among colony morphology and growth performance（P>0.05）;the results of proteolytic activities showed that gene deletions reduced the proteolytic activity of GL2significantly（P<0.05）;the results of hemolytic activities showed that deletion in alone only narrowed hemolytic circles slightly without significant differences（P>0.05）,while the deletion of both genes reduced its hemolytic activity significantly（P<0.05）;the results of motility assay and flagella formation displayed that gene deletions could not induce inability to synthesize flagella and significant decreases in the motilities of GL2（P>0.05）;besides,biofilm formation assay showed that gene deletions could inhibit the abilities of GL2 significantly（P<0.05）.3)In order to investigate the effects of gene deletions on GL2 infection to H.cumingii,challenge tests were set.The determination of median lethal dose(LD₅₀)showed that the LD₅₀ values ofΔPmb A andΔTld D were 5.89×10⁶ CFU/g,3.72×10⁶ CFU/g respectively,and no distinct differences were observed compared with GL2（1.98×10⁶ CFU/g）.Besides,the LD₅₀ value ofΔP&T was 1.25×10⁷ CFU/g,6.31 times as high as GL2.After challenging H.cumingii with wild-type GL2 and all mutant strains,the activities of immune-related enzymes in hemolymph,hepatopancreas,and gills exhibited different changes.The peak values of enzymatic activities inΔP&T group appeared at 12 h post infection basically,while GL2 group reached its peak values earlier and the emzymatic activities were significantly higher than other groups（P<0.05）;the histopathological observation of hepatopancreas and gills at 48 h post infection were conducted,and there existed different degrees of weakening trend of lesions inΔP&T group;q PCR was used to analyze the expression levels of immune-related genes in the hepatopancreas and gills of H.cumingii,and the results showed that the m RNA expression levels of Hc IL-17,Gal R2,s HSPs and TR in some mutant groups at 12 h post infection were significantly increased（P<0.05）.4)In order to further clarify the virulence mechanisms of genes pmbA and tldD in GL2,this study explored the metalloprotease-related pathways preliminarily.The mechanisms of proteolysis in mutant strains on different sources were explored,it was found that the peptidolytic activities of all mutant strains were significantly reduced（P<0.05）,but no significant changes were observed in caseinolytic activities（P>0.05）;in addition,the m RNA expression levels of genes pmbA and tldD were increased as higher concentrations of Na Cl;the m RNA expression levels in the potential synthesis pathways of metalloproteases Pmb A and Tld D were analyzed by q PCR,it was found that pmbA and tldD appeared to compensate for each other’s mutagensis,in which the deletion of one gene could upregulate the m RNA expression level of another gene.Besides,the m RNA expression levels of all mutant strains were decreased significantly（P<0.05）,and the deletion of both genes could downregulate the m RNA expression of aer A significantly（P<0.05）.To sum up,this study aimed to explore the virulence mechanisms by knocking out the metalloprotease genes pmbA and tldD in A.veronii GL2.pmbA and tldD could affect the proteolytic activities of GL2 by regulating its peptidolytic activity,and the hemolytic activity of GL2 might be affected by regulating its m RNA expression level of aer A.The deletion of both genes could significantly weaken the infectious ability of GL2 to H.cumingii.In addition,there might exist a compensate mechanism between genes pmbA and tldD,resulting in the deletion of one gene could not decrease the virulence of GL2 significantly.Therefore,the above results could provide references for subsequent researches on metalloproteases and other important virulence factors of A.veronii.