Construction Of PrkA Gene Deletion Strain Of Aeromonas Veronii And Preliminary Study On Its Function

Aeromonas veronii（A.veronii）is widely found in a variety of water sources.It not only causes huge economic losses to aquaculture,but also poses a serious threat to the health of humans,animals and aquatic organisms.A.veronii involves many virulence factors during host infection,such as outer membrane proteins,motility and adhesion related factors,and effectors secreted by various secretion systems.At present,the research on the function of pathogenic regulatory proteins has become one of the important directions in the study of pathogenic mechanism.Among them,the specific functions of regulatory proteins and their regulatory pathways and molecular mechanisms have also become the main contents of research.Studies have shown that Hanks-type Ser/Thr kinases（STKs）present in bacteria can regulate a variety of bacterial physiological activities by mediating phosphorylation modification.STKs can interact with transcriptional regulators to regulate transcription and protein biosynthesis.It plays an important role in the biological processes of bacterial cell wall synthesis,division,motility,metabolism and virulence.Based on previous laboratory studies,genes encoding STKs,such as prk A gene,also exist in the genome of A.veronii.However,the function of A.veronii STKs is still unclear,so strengthening the study of A.veronii Hanks STKs will help to clarify its specific function,provide a basis for further elucidating the pathogenic mechanism of A.veronii,and provide new research ideas for developing effective prevention and control strategies.In this study,homologous recombination was used to construct a deletion strain△prk A and its complement strain C-prk A of the gene prk A encoded by A.veronii TH0426 Hanks-type STKs Prk A.Then,the biological phenotype changes were analyzed to preliminarily clarify the function of Prk A.The results showed that deletion strain△prk A and its complement strain C-prk A were successfully constructed at genome,m RNA and protein levels.Drug susceptibility test showed that△prk A changed from sensitive to cephalexin and clindamycin to moderately sensitive,while the sensitivity of△prk A to other common antibiotics did not change.Moreover,compared with the wild-type strain A.veronii,the swimming ability of△prk A was enhanced,but the difference was not significant.The results of temperature tolerance test showed that compared with the wild strain A.veronii TH0426 and the complement strain C-prk A,the colony of deletion strain△prk A was larger after high temperature treatment at 50℃for 1 h,and the viable count of△prk A deletion strain△prk A was significantly increased compared with the wild-type strain（p<0.001）.The results of the adhesion ability assay showed that the adhesion rate of the deletion strain△prk A to Epithelioma Papulosum Cyprini（EPC）cells was 8.1±0.7%,while that of the wild-type A.veronii TH0426 was 5.7±0.8%.The adhesion ability of the deletion strain△prk A was significantly enhanced（p<0.05）.The effect of deletion of the prk A gene on the virulence of A.veronii was further analyzed at the cellular level and at the animal level,and the results of the cytotoxicity assay showed that at 2 h of infection,the deletion strain△prk A produced 1.66 times more toxicity to EPC cells than the wild-type strain,with a significant difference（p<0.05）.At 3 h of infection,the deletion strain△prk A produced 1.58times more toxicity to EPC cells than the wild-type strain,a highly significant difference（p<0.01）.The results of animal experiments showed that the LD₅₀of the deletion strain△prk A to zebrafish decreased by 2.51 times compared with that of the wild-type strain,and the virulence of the deletion strain△prk A was significantly enhanced（p<0.05）.To further analyze the mechanism by which the prk A gene regulates phenotypic changes in A.veronii TH0426,the differences between the deletion strain△prk A and the wild-type strain A.veronii TH0426 at the transcriptional level were comparatively analyzed by transcriptome sequencing（RNA-seq）.RNA-seq analysis showed that there were 984 differentially expressed genes（DEGs）between the deletion strain△prk A and the wild-type strain A.veronii TH0426,of which 572 DEGs were up-regulated and 412 DEGs were down-regulated.These DEGs were enriched to 70 KEGG metabolic pathways,mainly involved in metabolic pathways such as bacterial ribosome,flagellar assembly,T2SS,and lipopolysaccharide biosynthesis.The expression levels of some genes in related metabolic pathways were further verified by RT-q PCR.The results showed that prk A was involved in the regulation of metabolic pathways such as temperature tolerance and virulence of A.veronii.Through mediating the expression of lpx T,fli H,exe E,dna K,rpl C,mrc A and other DEGs in the pathway,the phenotypes of A.veronii such as drug sensitivity,temperature tolerance and virulence were regulated.In summary,the results of this study suggest that A.veronii TH0426 Hanks-type STKs Prk A is an important global regulator that negatively regulates several biological processes such as temperature tolerance,flagellar assembly and virulence in A.veronii.The results of the study provide a theoretical basis for further revealing the function of A.veronii TH0426 Hanks-type STKs Prk A and an important reference for further elucidating the pathogenic mechanism of A.veronii.