| Aeromonas veronii(A.veronii)is a new type of pathogen that can cause diseases in humans,animals,and fish.Its pathogenic mechanism is not fully understood.Hfq is an RNA-binding protein that assists small RNA(also known as sRNA)to mediate post-transcriptional regulation of genes,and plays a global regulatory role.Existing studies have shown that Hfq plays an important role in the pathogenicity and virulence of A.veronii.So this study took hfq deletion strain as the research object and sought Hfq-mediated signal pathway changes in order to better understand the function of Hfq in the regulation of A.veronii pathogenicity.First,multi-omics analysis of transcriptomics and metabolomics were found that the deletion of hfq led to the up-regulation of the Leucine biosynthesis-related gene ilvD,the incease of the concentration of Leucine in bacteria,and the defectived biofilm formation.Exogenous addition of 10 mM Leucine proved that the accumulation of Leucine has inhibitory activity on biofilm formation.Secondly,a new type of sRNAN155 that was downreguated in response to hfq deletion was discovered through transcriptome analysis,and the sRNAN155 knockout strain was constructed by homologous double exchange,which proved that the sRNA has an impact on bacterial membrane formation,movement and oxidation tolerance.And the phenotype is consistent with the hfq deletion strain.Finally,by constructing an eGFP fusion expression vector and using double plasmid transformation and fluorescence detection method,it was proved that sRNA N155 has a negative regulatory effect on the expression of ilvD.With point mutations combined with compensatory mutation verification experiments,it was proved that sRNA N155 takes the action of regulating the transcription level of ilvD gene by binding to ilvD at the coding region of 1358 bp-1382 bp,most importantly at the region of 1366-1372 bp.Furthermore,the membrane-forming ability and mobility of hfq deletion strain and sRNA N155 deletion strain were detected by exogenous addition of 10 mM Leucine,which confirmed that the presence of Leucine would enhance the inhibition of bacterial biofilm formation and mobility.Based on the results of this study,we propose the molecular mechanism by which Hfq assists sRNAN155 for mediating the regulations on Leucine metabolism and thus on biofilm formation.In this mechanism,Hfq acts as an RNA chaperone to assist sRNAN155 in the post-transcriptional regulation of ilvD,deletion of hfq leads to defective regulation of sRNAN155 and the transcription level of ilvD is up-regulated,which causes the accumulation of Leucine in bacteria,thereby inhibiting the ability of biofilm formation.This research is of great significance for discovering new pathogenic targets and designing and synthesizing new drugs,and can provide new ideas and methods for the study of the pathogenic mechanism of A.veronii. |
