Molecular Mechanism Of ZupT And ZnuABC Regulating The Virulence Of Mesophilic Aeromonas Salmonicida SRW-OG1

Aeromonas salmonicida,which can cause furunculosis in a wide range of freshwater and marine fish,causing serious economic losses to the global aquaculture industry.In this study,the pathogenic strain isolated from the diseased Epinephelus coioides in Zhangzhou,Fujian Province was named as SRW-OG1 and used as the research object,while the species of the pathogenic strain was confirmed by 16S rDNA.Whole genome sequencing and RNA-seq analysis were performed on this strain to study the mesophilic virulence of A.salmonicida.According to the joint analysis of genome and transcriptome,the zupT and znuABC which encoding zinc transporters were selected,and the effects of Zup T and ZnuABC on zinc acquisition and pathogenicity of the SRW-OG1 were explored.The main research contents are as follows:1)In this study,the pathogenic strain SRW-OG1 was isolated and cultured from E.coioides with furunculosis,and it was identified as A.salmonicida by 16S rDNA.By scanning electron microscopy to observe the shape characteristics of the pathogenic strain and observe the colony morphology on the LB ager plate,we found that it was basically the same as the biological characteristics of other A.salmonicida,that is,short rod-shaped,with flagellar,forming light yellow,smooth round colonies.Artificial infected E.coioides displayed the typical symptoms of diseased fish such as lumps on both sides of the body in the early stage,followed by ulcers,scabies,bleeding at the base of the fins,and bleeding points in the liver and other organs after later dissection.At the same time,the LD₅₀ was measured to be 2.1×10⁵ CFU/m L.Subsequently,the pathogenic strain was cultured at 18°C and 37°C for 24 h to detect the growth capacity,and we found that the strain could grow normally at both 18°C and 37°C.Finally,the indole test was performed on the pathogenic strain,and the result showed a clear chromogenic ring,indicating that the strain has the ability to metabolize tryptophan.2)In present study,the first whole genome sequencing study of the A.salmonicida SRW-OG1 was carried out,9,461 genes with biological functions were identified.Among them,6,250genes can be classified into 26 functional categories of the COG family.Meanwhile,6 antibiotic resistance genes and 12 prephages were identified,indicating that the A.salmonicida SRW-OG1may have acquired some antibiotic resistance and enhanced the environmental adaptability of the SRW-OG1 strain.Also,1,035 pathogen-host interaction factors and 566 virulence factors were identified in the SRW-OG1 strain,and compared with the two mesophilic strains,we found that they shared 276 pathogenic-host interaction factors,which is a proof that A.salmonicida SRW-OG1 is pathogenic in mammals and humans.3)In our study,the differential expression genes of A.salmonicida SRW-OG1 were analyzed at different temperatures,2,784 temperature-dependent differential expression genes were identified,which were candidates for investigating the mesophilic pathogenicity in this strain.Compared with the 18°C group,110 genes were significantly downregulated and 175 genes were significantly upregulated in the 37°C group,418 genes were significantly down-regulated and 45genes were significantly upregulated in the 28°C group.The functions of“Bacterial chemotaxis”and“ABC transporters”were significantly affected at 28°C compared with the 18°C group.At37°C,the functions of“Salmonella infection”and“Pathogenic E.coli infection”were significantly affected.4)The silent strain of A.salmonicida SRW-OG1 zupT gene was constructed by RNAi using,and the virulence regulation,zinc uptake and environmental adaptability of the silent strain were evaluated.Compared with the wild type,zupT-RNAi were severely restricted by growth under zinc and iron restriction,and the biofilm formation capacity,motility capacity,adhesion ability in vitro and hemolytic activity were significantly reduced.The mortality rate of zupT-RNAi infected E.coioides decreased by 80%,indicating that zupT gene was an important virulence regulator of A.salmonicida SRW-OG1.5)znuABC silent strains of A.salmonicida SRW-OG1 were constructed by RNAi using,while the zinc uptake and environmental adaptability of the silent strains were discussed.Compared to the wild type,znuABC silencing strains were not restricted in zinc limited environment,but their growth was clearly inhibited in iron restricted environment.The biofilm formation ability,motility capacity,adhesion ability in vitro and hemolytic activity of silent strains were significantly reduced.In a variety of stresses environment,the response of znuABC to the environment were not consistent,but the expression levels of znuABC increased significantly in zinc and iron restricted environments.This indicates that znuABC genes play an important role in the regulation of zinc acquisition and virulence regulation of A.salmonicida SRW-OG1.