Construction And Characterization Of Fur Deleted Mutant In Aeromonas Veronii TH0426

Aeromonas veronii(A.veronii)is a pathogenic bacterium of the genus Aeromonas that is widespread in the aquatic environment and can cause diseases such as meningitis and septicaemia in mammals,including humans,as well as fin rot in aquatic organisms.Reports indicate that the virulence of A.veronii is increasing and that cases of disease caused by this bacterium are becoming more frequent.It is important to study the genes associated with the virulence of A.veronii in order to better explore and control this bacterium.The Fur gene plays a key role in the bacterium as an iron uptake regulatory protein.Fur also acts as a global regulator,regulating not only iron-related expression,but also non-iron related pathways.The genomic analysis of the three types of A.veronii strains,namely A.veronii strong,weak and non-virulent,revealed that A.veronii TH0426 is a unique iron uptake regulator compared to other weak strains,and it is assumed that it acts as a virulence factor to determine the virulence of A.veronii.In order to investigate the function of the A.veronii Fur gene and its pathogenicity,the recombinant suicide plasmid p PE112-UDFur was constructed and the deletion strain ΔFur was successfully constructed by homologous recombination.The results showed that the growth ability of ΔFur was slightly lower than that of the wild strain after Fur deletion,and its resistance to antibiotics did not change significantly;the flagellum was broken and its swimming ability was significantly reduced by transmission electron microscopy;the morphology of some of the organisms was different by scanning electron microscopy,and the cross-linked structure of the biofilm became loose and stained with crystalline violet.Analysis supported the findings of a significant reduction in biofilm;the deletion strain ΔFur showed a significant reduction in adhesion and invasion of Epithelioma papillosum cyprinid(EPC)cells and reduced cytotoxicity,with a 2.3-fold reduction in the pathogenicity of ΔFur in zebrafish attack assays.To investigate the regulatory mechanism of Fur gene,transcriptome sequencing analysis of ΔFur was performed using the wild strain as a control group,and a total of 2242 differential genes were screened,including 993 up-regulated genes and 1249 down-regulated genes.The expression of the Fur gene was significantly different,and the phenomena generated by the biological characterization were consistent with the transcriptome results.In this study,the A.veronii Fur gene deletion strain ΔFur and the backfill strain C-Fur were successfully constructed and compared with the wild strain.The deletion of the Fur gene revealed a significant reduction in virulence,adhesion and invasion ability to EPC cells,bacterial own motility,biofilm formation and pathogenicity.This indicates that the Fur gene plays an important role as a global regulator of A.veronii and has contributed to the exploration of the pathogenesis of A.veronii,with a view to providing a theoretical basis for the control of A.veronii and the development of its vaccine.