Functional Study Of Small Non-coding RNA SR082 And SR012 Of Edwardsiella Piscicida

Edwardsiella piscicida is a major pathogen of marine fish in aquaculture.Edwardsiellosis caused by E.piscicida leading to severe economic loss.In recent years,Small noncoding RNA(sRNA)of bacteria function as regulators of gene expression at the post-transcriptional level and play critical regulatory roles in major biological processes,such as adaptation to various environmental stresses,quorum sensing,bioflm formation,motility,and pathogenicity.The infection mechanism of E.piscicida is not to be fully understood.Especially,the regulatory roles of small non-coding RNA(sRNA)is almost unknown in the pathogenicity of bacterium.In this study,total RNA was extracted from E.piscicida grown in normal media(Con),acidic stress(Ac),iron defciency(Dp)and oxidation pressure(Pe)and subjected to RNA deep sequencing.A total of 148 sRNAs were found,including 19 previously annotated sRNAs and 129 novel sRNA.Compared in normal condition,the expression of 103 sRNAs showed signifcant diferences in three stress sample.Based on these results,sRNAs with differential expression under acidic and oxidative conditions(named sR082 and sR012)were selected for further study.Firstly,the expression levels of sR082 and sR012 in different growth phase of E.piscicida under various conditions were detected by qRT-PCR.The results showed that sR082 was highly expressed at early phase of bacterial growth and low pH conditions.And sR012 was highly expressed at logarithmic phase.To analyze the functions of sR082 and sR012,we constructed knockout strains TXΔsR082 and TXΔsR012 by In-frame deletion.The growth of knockout strains TXΔsR082 and TXΔsR012 and wild strains TXD1 was compared under low pH pressures and oxidative stress conditions.The results showed that the growth rate of TXΔsR082 significantly reduced under pH 5 conditions and the TXΔsR012 significantly decreased at entire growth phase compared with the wild-type strain under oxidative stress.These results indicated that sR082 and sR012 was related to the ability of bacteria to adapt to acid and oxidative stress environments,respectively.To further analyze the effects of sR082 and sR012 on infection hosts,our study analyzed the virulence of TXΔsR082,TXΔsR012 and TXD1 for cell line,tissues and individuals,respectively.The results showed that compared with TXD1,the mutant strain TXΔsR082 and TXΔsR012 exhibited significantly reduction on infecting and replicating in flounder FG cells,dissemination in tissues,and lethality to fish.These observations suggest that sR082 and sR012 plays important roles in response to acid and oxidative stress and virulence of E.piscicida.To further understand the role of sR012 in E.piscicida,we used TargetRNA2 to predict the sR012 target gene and performed qRT-PCR assay to detect the expression level of the putative target gene.The results showed that the total of four target genes(ETAE-0114,ETAE-2575,ETAE-3156 and ETAE-0911)were differentially expressed in TXΔsR012,and sR012 may regulate these four target genes to affect capacities of adaption oxidative stress and bacterial virulence.