The Molecular Characteristics And Functional Research Of AsRNA AsfD In Salmonella Enterica Serover Typhi

Objective:Bacterial non-coding RNAs?ncRNAs?can regulate a lot of bacterial processes such as motility,biofilm formation and virulence.High-throughput sequencing and transcriptome analysis of the Salmonella enterica serovar Typhi?S.Typhi?found a lot of ncRNAs.One of these ncRNAs encoded from the opposite strand of the flhDC operon attracted our attention,which was named as AsfD.In this study,we want to identify the molecular characteristics of the Asf D,and investigate the expressional characterization,functions and regulatory mechanism of the AsfD.Methods:1.Molecular characteristics of AsfD.The specific probes were designed in the known sequence region of AsfD.Then,RT-PCR and Northern blot was used to confirm the expression of this antisense RNA AsfD in S.Typhi.Using 5'RACE and RT-PCR,we determined the transcription initiation site and probable termination area of AsfD,in combining with Northern blot to analyse the full length of the AsfD.2.Analysing the expression characteristics of AsfD.Northern blot and qRT-PCR assay were used to analyze expressional levels of AsfD in different growth phases.qRT-PCR was used to analyze the expression characteristics of AsfD in different stress conditions?acid,oxygen,hypertonic?.3.Construction of over-expressing strain of AsfD.According to the results of Northern blot,5'RACE and RT-PCR assay,a pair of specific primers of AsfD were designed for PCR.The PCR fragment includes Noc I and EcoR I restriction endonuclease sites,and was cloned into the pBAD/HisA to construct the AsfD over-expressing strain?WT+pBAD-asfD?and the control strain?WT+p BAD?.4.Motility assays.AsfD over-expressing strain and control strain were inoculated into a semi-solid medium and cultured overnight to measure the diameter of the S.Typhi motility circle.5.Biofilm formation assays.AsfD over-expressing strain and control strain were grown intrypticase soy broth?TSB?to OD₆₀₀00 0.4.These two strains were incubated into 96-well microtitre plates.After 96 h,crystal violet was used to dye the biofilm.The absorbance of the eluted stain was measured at 570 nm to quantify biofilm biomass.6.The epithelial cells invasion assay.AsfD over-expressing strain and control strain were grown to log-phase(OD₆₀₀00 0.4).The strains were inoculated into 24-well plate,in which HeLa cells had been incubated.The invasion ability between the two strains were then compared.7.Detection of target gene flhDC and flagella genes using qRT-PCR.AsfD over-expression strain and the control strain were grown to log-phase and then induced with 0.2%?w/v?arabinose for 30min.Total RNAs were isolated using TRIzol.qRT-PCR was used to detect the expression of flhDC,fliA and fljB.8.Detection of FljB protein level using Western blot.AsfD over-expression strain and control strain were grown until log-phase and treated with 0.2%?w/v?arabinose for 0,15,30 and 60 min.Bacteria extracts were prepared by sonication.Western blot was used to detect the differences in AsfD over-expression strain and control strain.Results:1.The results of Northern blot revealed a full-length transcript approximately 2300 nt.The result of 5'RACE revealed the 5?-end of the transcript was 590 bp downstream of the termination codon of flhC gene.The result of RT-PCR revealed 3?-end of the transcript was 558 bp⁷88 bp upstream of start codon of flhD gene.2.The results of Northern blot and RT-PCR revealed that the AsfD expression increased along with bacterial growth and reached the highest level in the stationary phase.The level of Asf D decreased under acidic,oxidative and hyperosmotic stress.3.The over-expression strain?WT+pBAD-asfD?and control strain?WT+pBAD?were successfully constructed.4.The results of motility tests showed that the motility of AsfD over-expression strain was significantly enhanced compared with the control strain.5.The results of the biofilm formation assays revealed that the biofilm formation of AsfD over-expression strain was enhanced compared with the control strain.6.The results of the epithelial cells invasion assay showed no significant difference between AsfD over-expression strain and control strain.7.The results of the qRT-PCR revealed showed that the AsfD over-expression strain may up-regulate expression of flhDC,fliA and fljB compared with the control strain.8.The Western blot assay results revealed that the AsfD over-expression strain may up-regulate expression of FljB compared with the control strain.Conclusions:In this study,A long asRNA AsfD was confirmed in S.Typhi with a full length of2079 nt²310 nt.AsfD,which has been identified as 2079 nt sequence,can completely cover flhDC operon and may enhance biofilm formation.Asf D may also enhance motility by directly up-regulating expression of the target gene flhDC in S.Typhi.