| Aeromonas veronii(A.veronii)is a wide host of Gram-negative pathogens,which can infect a variety of economic fish,bring irreparable economic losses to aquaculture,and also infect a variety of mammals including humans,threatening public health and safety.At present,although a variety of virulence factors of A.veronii have been identified,the pathogenic mechanism of this pathogen is not clear,which is also an important reason for limiting us to formulate effective environmental protection control measures.Two-Component systems,TCSs)is the main signal sensing and conducting system in prokaryotes.Qse B/Qse C is composed of Qse C histidine kinase(HK)and Qse B response regulator(RR),which has been proved to be involved in the transcriptional regulation of virulence-related genes of many pathogenic bacteria.However,there is no report on the two-component function of A.veronii Qse BC at present.Therefore,exploring the function of Qse BC in A.veronii will help us to further understand the pathogenic mechanism of A.veronii.In this study,the wild strains TH0426 isolated in the early laboratory was used as the reference control strain,and the qse B,qse C,and qse BC gene deletion strains and complementary strains of A.veronii were constructed by homologous recombination,and the biological phenotypic changes were compared and analyzed to identify the correlation between the two-component Qse BC and the virulence regulation of A.veronii,We also explored the related metabolic pathways in which Qse B,Qse C,and Qse BC were involved in the regulation of A.veronii.Finally,we screened the target genes directly combined with Qse B by using Ch IP-Seq to explore the molecular mechanism of two-component Qse BC in the regulation of A.veronii.The main research contents are as follows:(1)Constructions of qse B,qse C and qse BC gene mutant strains and complementary strains of Aeromonas veroniiIn this study,the Qse BC two-component coding sequence of A.veronii was found by using the previous genome annotation results of A.veronii strain TH0426.The suicide plasmid p RE112was selected to construct homologous recombination vectors p RE112-qse B,p RE112-qse C and p RE112-qse BC,which were transferred into the wild TH0426 by natural transformation.After screening by Amp~+and Cm~+resistance plates.The mutant strainsΔqse B,Δqse C andΔqse BC were obtained.At the same time,the promoter and ORF sequences of the two-component gene of Qse BC were found.After PCR amplification,they were connected to the p EASY cloning vector in series,and then connected to the wide-host expression plasmid p BBR-MCS1 to construct recombinant plasmids p BBR-qse B,p BBR-qse C and p BBR-qse BC.Similarly,the recombinant plasmids were transformed into the mutant strainsΔqse B,Δqse C andΔqse BC by natural transformation method.After screening with Amp~+and Cm~+resistance plates,the complementary strains C-qse B,C-qse C and C-qse BC were obtained.The successful construction of gene mutant strain and complementary strain was verified by DNA and RNA levels.(2)Analysis of biological characteristic of wild strains,missing strain and complementary strainIn this study,the biological characteristics of wild TH0426;mutant strainsΔqse B,Δqse C andΔqse BC;and complementary strains C-qse B,C-qse C and C-qse BC were analyzed comparatively.The results of colony morphology and growth curves showed that the mutant of qse B,qse C or qse BC genes did not change the colony morphology,but the deletion of qse B gene significantly reduced the growth rate of A.veronii in log phase;the results of motility assay showed that the deletion of qse B,qse C and qse BC genes could significantly reduce the growth rate of A.veronii in log phase.The results showed that the mutant of qse B,qse C and qse BC genes significantly reduced the swimming ability of A.veronii,and the flagellum staining and transmission electron microscopy showed that the deletion of qse B and qse C genes significantly enhanced the biofilm formation of A.veronii.The results showed that deletion of the qse B and qse C genes significantly enhanced the biofilm formation of A.veronii,and a large amount of biofilm formation ofΔqse B andΔqse C was also observed in the scanning electron microscope field.The results showed thatΔqse B andΔqse C were 1.24-fold and 1.42-fold higher,respectively,than the wild strain,whileΔqse BC was 1.83-fold lower than the wild strain;and the cytotoxicity assay showed thatΔqse B andΔqse C were 1.24-fold higher and 1.42-fold lower,respectively,than the wild strain.At 30 min of infection,the toxicity of the deletion strainsΔqse B,Δqse C andΔqse BC to EPC cells decreased4.17-fold,3.02-fold and 3.21-fold,respectively,compared with the wild strain;at 1 h of infection,they decreased 2.36-fold,2.02-fold and 2.46-fold,respectively,compared with the wild strain;at2 h of infection,they decreased 1.28-fold,0.87-fold and 1.49-fold,respectively,compared with the wild strain.Similarly,the results of the zebrafish pathogenicity test showed that the LD50 ofΔqse B,Δqse C andΔqse BC were 74,8.5 and 61 times higher than that of the wild strain TH0426,respectively,indicating that the Qse BC doublet was involved in the regulation of the pathogenicity of A.veronii,and it seems that in The results of the bacterial load test showed that the bacterial load in the blood,liver,spleen and kidney of the deletion strain was significantly lower than that of the wild strain;when the infection reached 24 h and 48 h,the deletion strainΔqse BC had the lowest fixed values in all tissues;the results of the in vivo competition test showed that all competition indices were less than 1,indicating that Qse BC The results of in vivo competition tests showed that all competition indices were less than 1,indicating that the two components of Qse BC could promote the interbacterial competition ability of A.veronii in the host.(3)Comparative transcriptomic study of wild-type and mutant strainsTo further elucidate the underlying causes of the phenotypic changes in A.veronii after mutant of qse B,qse C and qse BC genes,we performed a comparative transcriptomic study of the deletion strainΔqse B,Δqse C andΔqse BC with the wild-type TH0426 strain using RNA-Seq technology.Compared with the wild-type TH0426 strain,924 differentially expressed genes were present in the mutant strainΔqse B,containing 633 up-regulated and 291 down-regulated expressions;2364 differentially expressed genes were present in the mutant strainΔqse C,containing 1141 up-regulated and 1223 down-regulated expressions;152 differentially expressed genes were present in the mutant strainΔqse BC,containing 152 differentially expressed genes in the mutant strainΔqse BC,including 129 up-regulated expressions and 23 down-regulated expressions.Bioinformatics analysis showed that Qse BC was involved in the regulation of metabolic pathways such as growth performance,chemotaxis,secretion system,flagellar assembly,and biofilm formation in A.veronii,and was consistent with the phenotypes of A.veronii such as flagellar shedding,increased biofilm formation,and reduced virulence,indicating that the Qse BC double component plays an important regulatory role in the pathogenicity of A.veronii.The Qse BC dual component plays an important regulatory role in the pathogenicity of A.veronii.(4)Ch IP-Seq analysis Qse B target geneIn order to further clarify the regulatory mechanism of Qse BC on A.veronii,this study used Ch IP-Seq technology to screen potential target genes regulated by Qse B,and the key to this technology is the need for high quality Ch IP-grade antibodies.Therefore,we combined the qse B gene(qse B promoter)with the coding sequence of qse B gene and Flag tag sequences into p BBR-MCS2 vector to construct a recombinant expression plasmid p BBR-MCS-qse B-flag,and electrotransferred into the mutant strainΔqse B strain,and verified by RT-q PCR and Western-blot that the recombinant plasmid could be stably expressed in the deletion strainΔqse B strain;by Ch IP-Seq,we screened 1639 Pea K,containing 1291 genes,and after combined RNA-Seq and Ch IP-Seq analysis,347 potentially regulated target genes of Qse B were screened,including 24genes fundamentally related to phenotypic changes,namely,bacterial chemotaxis-related:che Y;flagellar assembly-related:flg F,flg G,flg M,flg H,flg E,flg L,fli J,fli F,fli O,fli L,fli B;biofilm formation related:glg C,omp A,pts G,crr;sec translocation system:sec G,sec Y,sec A;signal recognition protein particle:ffh;bidirectional arginine targeting system:tat B;type IV secretion system:vgr G,lip,hcp.Among them,Qse B is able to bind to the promoter region of che Y and omp A genes.(5)Qse B regulates the expression of che Y and omp A genesQse B,as a two-component response regulator of Qse BC,can not only control gene expression by binding to promoters,but also directly modify the functional activity of target proteins,thus regulating downstream responses.After combined RNA-Seq and Ch IP-Seq analysis,it was found that che Y and omp A might be potential target genes of Qse B,and it was demonstrated by EMSA assay that Qse B could directly bind to the promoters of che Y and omp A genes;in order to further clarify the regulatory relationship of Qse B on che Y and omp A,this study used p BBR-lacz reporter.To further clarify the regulatory relationship between Qse B and che Y and omp A,the p BBR-lacz reporter plasmid was used for verification.In order to eliminate the experimental background of lacz gene production in the strain,the lacz gene deletion strainsΔlacz andΔqse B-lacz of wild strain TH0426 and deletion strainΔqse B were constructed in this study,respectively.In summary,this study revealed that Qse BC is involved in the regulation of growth division,chemotaxis,biofilm formation,flagellar assembly and virulence of A.veronii,and the downstream regulation of Qse B by che Y and omp A.The results also provide an important basis for further elucidation of the pathogenesis of A.veronii. |
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