The Construction And Functional Analysis Of QseC Gene Deletion Mutant In Aeromonas Hydrophila

Aeromonas hydrophila is a conditional pathogen which is widespread in water.Both freshwater farmed fish and wild fish can be infected,causing acute hemorrhagic septicemia,resulting in a large number of deaths and huge economic losses.Stress is an inevitable factor in aquaculture industry and an important trigger for outbreaks of pathogenic bacteria from aquatic animals.In response to stress,the host produces a large amount of catecholamine hormones,such as norepinephrine(NE),epinephrine(Epi).Pathogens can recognize these environmental signal factors,promote bacterial growth,and enhance bacterial virulence.The QseBC two-component regulatory system can regulate the expression of related virulence genes through the receptor protein QseC sensing signal molecules Epi/NE,and carry out the information exchange between bacteria and host.Studies have shown that NE can promote the growth of A.hydrophila NJ-35 and enhance bacterial virulence,but the specific regulatory mechanism is unclear.Aeromonas hydrophila NJ-35 contains a gene encoding the QseBC two-component regulatory system,while whether this system is involved in the recognition and response to NE still needs further study.In order to determine whether QseC can recognize NE and participate in the regulation of Aeromonas hydrophila toxicity,this study constructed a mutant strain AqseC,and carried out the related research on its in vivo and in vitro toxicity in the presence of NE and the results are as follows:In vitro experiments,the absence of qseC inhibited the growth-promoting effect of NE on Aeromonas hydrophila;during NE exposure,the hemolytic activity and biofilm formation ability of ΔqseC were significantly reduced(P<0.01),compared with the wild-type strain NJ-35.This result indicated that QseC may affect the pathway of which NE regulated the hemolytic activity and biofilm formation of Aeromonas hydrophila NJ-35.In addition,after NE incubation,the adhesion of wild type strain to J774A.1 cells was significantly enhanced(P<0.01),while the adhesion of △qseC to cells was significantly reduced(P<0.01),indicating that QseC was involved the regulation of NE on the adhesion ability of Aeromonas hydrophila.However,there was no significant effect on the motility,lipaseactivity and protease activity between wild type strain and △qseC(P>0.05).The results of studies in vivo showed that the deletion of the qseC gene attenuated the effect of NE on enhancing the virulence of Aeromonas hydrophila,thereby reducing the oxidative stress and tissue damage suffered byCarassiusauratus gibelio.It is mainly manifested that under the induction of NE,compared with the fish injected with wild type strain NJ-35,the CAT activity of that injected with △qseC had enhanced as well as GSH,however the activities of ALT,AST,ALB and ALP were decreased(P<0.05).Furthermore,NE can dramatically increase the mortality of the wild type strain NJ-35 against C.gibelio,while the deletion of the qseC gene inhibited the enhanced-virulence by NE.These indicated that QseC could regulate the effect of NE on the pathogenicity of Aeromonas hydrophila.In summary,the deletion of the qseC gene inhibits the growth-promoting effect of NE on Aeromonas hydrophila NJ-35,and weakens the enhanced ability of NE to biofilm formation,hemolytic activity,and cell adhesion of Aeromonas hydrophila NJ-35.The data showed that QseC can regulate the pathogenicity of Aeromonas hydrophila NJ-35 by identifying and responding to NE.This study can further analyze the virulence regulation of Aeromonas hydrophila and the mechanism of interaction with its host,providing a theoretical basis for new technologies for its disease control strategies.