| Edwardsiella piscicida（E.piscicida）,a gram-negative bacterium,mainly infects various fish species,causing huge economic losses to the aquaculture industry.Two-Component System（TCS）is a widely used signal transduction System in bacteria,which can participate in gene regulatory network in response to environmental signals and play an important role in the process of pathogen infection.FusKR is an important TCS,and studies have shown that FusKR in Enterohemorrhagic can regulate its metabolism and pathogenicity by sensing fucose in the host intestinal tract,whereas.The main function of the E.piscicida FusKR is unclear.On the basis of the previous research,this study further explores FusKR in Escherichia coli（EHEC）.In order to elucidate the key protein and action pathway of FusKR and provide theoretical support for the prevention and control of E.piscicida.Previous studies have shown that the optimal concentration of L-fucose on the growth performance of E.piscicida is 30 mm.E.piscicida wild virus strain EIB202 was cultured in LB medium with and without 30 mm L-fucose,and RT-q PCR results showed that L-fucose promoted the fusK gene in the E.piscicida FusKR two-component system（The transcription of the gene encoding the kinase FusK）（P<0.05）and its regulated gene ETAE＿1977（encoding the fucose transporter Fuc P）（P<0.01）,inhibited the transcription of the fus R gene（encoding the regulatory protein Fus R）（P<0.01）.We constructed E.piscicida fusK gene deletion strain ?fusK and fus R gene deletion strain?fus R.After 50 passages of subculture,the deleted gene did not have reverse mutation,and?fusK and ?fus R were genetically stable;When ΔfusK and Δfus R were cultured on the medium,they still appeared as colonies with black center and translucent edge;the growth performance of EIB202,ΔfusK and Δfus R in LB medium had no difference（P>0.05）,and the fusK and fus R genes were not E.piscicida Δfus R entered the stable growth phase faster（P<0.05）;we detected EIB202,ΔfusK on 0.5% LB semi-solid medium.and Δfus R motility,the results showed that ΔfusK and Δfus R were smaller in diameter than EIB202 motility ring（P<0.01）,and deletion of both fusK and fus R genes attenuated the motility of E.piscicida;EIB202,ΔfusK and Δfus R were cultured in LB medium of alginose,and RT-q PCR analysis showed that the deletion of fusK gene inhibited the transcription of ETAE＿1977 and fus R genes（P<0.01）;the deletion of fus R gene promoted ETAE＿1977 and fusK genes transcription（P<0.01）.We constructed a His-tagged p ET28a-fusK fusion protein expression vector,induced expression,purified and verified it as a bait protein,incubated with the total protein of EIB202 strain,and "fished" the total protein of EIB202 by His pull-down test.or target proteins bound indirectly to FusK.A total of 1285 potential FusK-interacting target proteins were identified via LC-MS/MS mass spectrometry analysis.Furthermore,GO（Gene Ontology）functional analysis,COG（Cluster of Orthologous Groups）annotation and Pathway annotation were performed on these proteins.The results showed that many of the interacting proteins screened were related to the virulence of E.piscicida,such as type III secretion.System（T3SS）and type VI secretion system（T6SS）related proteins,as well as most metabolism-related proteins,such as glycerol-3-phosphate 1-O-acyltransferase Pls B（involved in oxidative stress resistance）,FusKR system regulates E.piscicida virulence and metabolism.Based on the analysis results of interacting proteins,we detected the biofilm formation ability,oxidative stress resistance ability,T3 SS effector gene（ese E）and T6 SS effector gene（evp J）transcription levels of EIB202,ΔfusK and Δfus R.The biofilm for ming ability of EIB202,ΔfusK and Δfus R was detected by quantitative（crystal violet staining method）and qualitative（DAPI staining method）at 6h,12 h and 24 h,respectively.The results showed that the biofilm for ming ability of ΔfusK and Δfus R was weaker than that of EIB202（P<0.01）,and the difference was most significant at 12 h,FusKR system enhanced the biofilm formation ability of E.piscicida;the results of oxidative stress resistance test showed that after EIB202,ΔfusK and Δfus R were treated with 10 mmol/L H2O2 for 15 min,the ΔfusK The survival rate was lower than that of EIB202（P<0.01）,the deletion of fusK gene weakened the oxidative stress resistance of E.piscicida,the survival rate of Δfus R was higher than that of EIB202（P<0.01）,and the deletion of fus R gene enhanced the resistance of E.piscicida Tolerance to oxidative stress.EIB202,ΔfusK and Δfus R were cultured in LB medium with and without 30 mm L-fucose,respectively,and the transcriptional differences of ese E and evp J genes were detected by q PCR,respectively.The results showed that L-fucose promoted the transcription of ese E gene（P<0.01）and evp J gene（P<0.01）;the deletion of fusK gene inhibited the transcription of ese E gene（P<0.01）and evp J gene（P<0.05）.The promoting effect of L-fucose on the transcription of ese E and evp J genes was stronger than the inhibition effect caused by the deletion of fusK gene（P<0.05）;the deletion of fus R gene inhibited the transcription of ese E gene（P<0.01）and evp J gene（P<0.05）.0.05),the promoting effect of L-fucose on the transcription of ese E gene was equivalent to the inhibition effect caused by the deletion of fus R gene（P>0.05）,and the promotion effect of L-fucose on the transcription of evp J gene was stronger than that caused by the deletion of fus R gene effect（P<0.05）.In conclusion,in the present study,we found that the novel two-component system FusKR of E.piscicida regulates its biofilm formation,resistance to oxidative stress,and important virulence factors（T3SS and T6SS）,which are involved in E.piscicida is resistant to resistance,resists the host i mmune system and causes great damage to it.We believe that the FusKR two-component system has the potential to develop new drugs as a target.New ideas and theories have important research significance. |
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