Preliminary Study On The Function Of Hanks-like Serine/Threonine Protein Kinase PrkC Of Aeromonas Veronii

Aeromonas veronii is an important zoonotic and aquatic agent,which can not only infect aquatic animals including fish,but also infect mammals including humans.It has caused substantial economic losses in the aquaculture industry,in addition to threatening human health.However,there is still a lack of effective prevention and control measures against A.veronii disease owing to little understanding of the pathogenic mechanism of A.veronii.Relevant studies have shown that bacterial Hanks-like serine/threonine protein kinase(eukaryotic-like serine/threonine protein kinase)is an important regulatory factor that can affect the pathogenicity of bacteria.At present,the majority of Hanks-like serine/threonine protein kinases are found in Gram-positive bacteria,and a few in Gram-negative bacteria,and there are significant differences in their functions in different bacteria.However,there is no report on the functions of A.veronii Hanks-like serine/threonine protein kinase.Therefore,in-depth study of its function will help us to further understand the pathogenesis of A.veronii.This study conducted a preliminary study on the functions of the two Hanks-like serine/threonine protein kinases Prk C and Yih E that exist in the genome of A.veronii TH0426 strain.First,the prkC and yih E gene deletion strains were constructed and the related biological phenotype were analyzed,and the functions of Prk C and Yih E were initially clarified.Then the related metabolic pathways that A.veronii Hanks-like serine/threonine protein kinase Prk C could regulate were found by comparative proteomics and analyzing the function of Prk C.Furthermore,the changes of A.veronii protein phosphorylation modification levels that before and after the prkC was deleted were comprehensively analyze using high-throughput phosphorylation quantitative proteomics technology.And the biological functions of phosphorylated differential proteins and the metabolic pathways they participate in were also analyzed.Finally,the Pull-down-MS technology was used to screen the target proteins that directly interact with Prk C protein,and to explore the molecular mechanism of Prk C-mediated phosphorylation to regulate the related phenotypic changes of A.veronii.The main findings are as follows:(1)The prkC and yih E mutant strains of A.veronii were successfully constructed,and the analysis found that the growth rate of the mutant strain?prkC during the logarithmic growth period was significantly lower than that of the wild strain,while the growth rate of the mutant strain?yih E was basically the same as that of the wild strain,indicating that the deletion of the prkC gene affects the growth rate of A.veronii.The adhesion ability test results show that the adhesion ability of the wild strain TH0426 to EPC cells is 6.73±0.5%,and the mutant strain?prkC is 2.63±0.4%,which has significant difference,and the mutant strain?yih E is 3.76±0.2%,there is also a significant difference,indicating that the deletion of prkC and yih E genes both weaken the adhesion of A.veronii to EPC,and the effect of prkC is more obvious than that of yih E.The toxicity test results showed that the toxicity of wild strain TH0426 to EPC was 6.54 times and 5.29 times than that of the mutant strain?prkC respectively after 2h and 3h of infection,the difference was very significant,and was 2.94 times and 1.64 times than that of the mutant strain?yih E with significant difference.It showed that the deletion of prkC and yih E genes weakened the toxicity of A.veronii to EPC cells in the early stage of infection,and compared with yih E,the effect of prkC was more obvious.The results of the zebrafish pathogenicity test showed that the LD₅₀ of the mutant strain?prkC was increased by2.04 times than that of the wild strain and its virulence decreased significantly,while the mutant strain?yih E increased by 1.44 times and the decreased change was not significant,which indicating that the deletion of prkC and yih E genes both weakened the pathogenicity of A.veronii to zebrafish.But compared to yih E,the effect of prkC is more obvious.In addition,the motility test results showed that the deletion of the prkC gene significantly affected the motility of A.veronii,and the flagella of the mutant strain?prkC was significantly shed,indicating that the loss of the prkC gene significantly affected the flagella formation of of A.veronii TH0426.(2)Comparative proteomics analysis results show that there are 314differentially expressed proteins between the mutant strain?prkC and the wild strain TH0426,of which 142 are up-regulated proteins and 172 are down-regulated proteins.The results of bioinformatics analysis show that the expression levels of the proteins related to metabolic pathways such as chemotaxis,flagella assembly,two-component system,bacterial secretion system,and biofilm formation of A.veronii were reduced.In addition,the deletion of prkC gene resulted in Mot A,Mot B,Fli M and Fli N and other 16 major flagella formation related proteins of A.veronii were significantly down-regulated,which was consistent with the phenotypic changes of the mutant strain?prkC,such as flagella shedding and reduced locomotor ability,indicating that the prkC gene is involved in regulating the flagella formation of A.veronii.(3)The results of phosphorylation modification analysis showed that there were75 different modification sites between the mutant strain?prkC and the wild strain TH0426,of which 56 were up-regulated modification sites,corresponding to 40proteins,and 19 were down-regulated modification sites,Corresponding to 17proteins.Furthermore,the functions of phosphorylated differential proteins were analyzed using bioinformatics method,it is found that serine/threonine protein kinase Prk C participates in many biological processes of A.veronii including transcription,translation,basic metabolism and bacterial chemotaxis through phosphorylation modification.And the analysis results show that Prk C participates in the regulation of A.veronii flagellin Fli C phosphorylation modification and protein expression.(4)8 proteins that have potential interaction with serine/threonine protein kinase Prk C include serine/threonine protein kinase,sodium ion translocation NADH-ubiquinone reductase sub Base A,bacterial flagellar filament protein Fli C,cell division protein Fts Z,glutamate-t RNA ligase Glt X,bacterial chemotactic protein Che V,citrate synthase Glt A and isocitrate dehydrogenase Icd were screened by Pull-down-MS technology.In addition,it is confirmed that there is a direct interaction between serine/threonine protein kinase Prk C and flagellin Fli C through in vitro Pull The-down experiment,indicating that Prk C directly acts on flagellin Fli C through phosphorylation modification,thereby affecting the flagella formation of A.veronii.In summary,the results of this study indicate that the serine/threonine protein kinase Prk C of A.veronii is an important global regulator,which participate in the regulation of multiple biological processes involved in growth and division,virulence,chemotaxis,flagella assembly and synthesis,etc.It laid the foundation for further clarifying the function of serine/threonine protein kinase Prk C of A.veronii,and also provided an important basis for further elucidating the pathogenesis of Aeromonas veronii.