| Objective: Non-coding RNAs(nc RNA)play an important role in response to environmental change and pathogenic processes.A new nc RNA,antisense RNA of bax(Asb),was discovered in the human pathogen Salmonella enterica serovar Typhi(S.Typhi)by deep sequencing analysis of transcriptome,and was identified as the 5’ untranslated region(5’UTR)of mal S gene,which was renamed as mal S-5’UTR.It may play an important role in S.Typhi colonization and intracellular survival in host macrophages.Therefore,this study identified the mechanism of action of mal S-5’UTR in macrophages to clarify the role nc RNA plays in regulation of environmental stress and pathogenesis of S.Typhi.Methods: 1.The construction of(35)pho P-mal S-5’UTR strain: The p BAD-mal S-5’UTR plasmid was electroporated into the pho P mutant strain to obtain the ?pho P-mal S-5’UTR strain by recombination.2.The macrophage intracellular survival assay: The WT-mal S-5’UTR,WT-p BAD and ?pho P-mal S-5’UTR strains were cultured in LB medium to the late stage of logarithmic growth phase and incubated with THP-1 cells.The survival ability of strains in macrophages were compared to evaluate the fuction of the mal S-5’UTR in S.Typhi.3.The growth curves assay in LPM: The WT-p BAD,WT-mal S-5’UTR and(35)pho P-mal S-5’UTR strains were incubated to the late logarithmic growth phase in LB medium,and then transferred into the LPM medium.The growth curves were drawn with the OD600 on Y-axis and the incubation time on X-axis to compare the growth condition between different strains in LPM medium.4.The genomic microarray assay: The WT-p BAD and WT-mal S-5’UTR strains were incubated in LPM medium for 12 h.Total RNAs were extracted,reverse transcribed into c DNA and reciprocally labeled with cy3-and cy5-d CTP simultaneously.After hybridization with the Salmonella genomic microarray,the scanning signals of fluorescence were digitized and used to analyze the gene expression differences between the two strains.In order to confirm the results of the microarray assay,changes observed in some genes in the microarray assay were selected for q RT-PCR analysis.5.Measurement of intracellular ATP in Salmonella: The intracellular ATP levels of the WT-p BAD,WT-mal S-5’UTR and(35)pho P-mal S-5’UTR strains were assayed with ATP content assay kits to compare the ATP levels between different strains.6.Construction of WT-mal S-5’UTR-Mgt C::3×Flag,WT-p BAD-Mgt C::3×Flag and(35)pho P-mal S-5’UTR-Mgt C::3×Flag fusion strains: The 3×Flag fusion with mgt C in the WT-mal S-5’UTR,WT-p BAD and(35)pho P-mal S-5’UTR were prepared by homologous recombination mediated by suicide plasmid.The 3×Flag tag was inserted directly before the termination codon of mgt C in the WT-mal S-5’UTR,WT-p BAD and(35)pho P-mal S-5’UTR to construct WT-mal S-5’UTR-Mgt C::3×Flag,WT-p BAD-Mgt C::3×Flag and(35)pho P-mal S-5’UTR-Mgt C::3×Flag fusion strains.7.Western Blot: WT-mal S-5’UTR-Mgt C::3×Flag fusion strain,WT-p BAD-Mgt C::3×Flag fusion strain and(35)pho P-mal S-5’UTR-Mgt C::3×Flag fusion strain were grown in LPM medium for 15 hours.Then bacterial proteins were harvested,and Western-blot analysis was used to detect the protein levels of Mgt C in the WT-mal S-5’UTR,WT-p BAD and(35)pho P-mal S-5’UTR with anti-FLAG.8.Analysis of the mechanism of mal S-5￠UTR regulation of the survival of S.Typhi in macrophage: The m RNA levels of pho P,pho Q,mgt C and atp operon were detected by q RT-PCR in the WT-mal S-5’UTR,WT-p BAD and(35)pho P-mal S-5’UTR strains.The above experiments were used to analyse the mechanism of mal S-5￠UTR regulation of the survival of S.Typhi in macrophage.Results: 1.The(35)pho P-mal S-5’UTR strain was constructed successfully.2.The macrophage intracellular survival assay indicated that overexpression of the mal S-5’UTR in S.Typhi weakened its ability to survive in macrophages.In the mutant pho P gene,the survival rate of the(35)pho P-mal S-5’UTR strain in macrophages was dramatically lower.3.The growth curve showed that the mal S-5’UTR obviously decreased the growth of S.Typhi when the strains were cultured in LPM medium.However,in the mutant pho P gene,the growth of the(35)pho P-mal S-5’UTR strain was significantly slower than that of WT-mal S-5’UTR strain.4.The genomic microarray assay revealed that 147 genes were expressed differentially in the WT-mal S-5’UTR and WT-p BAD strains.Among these genes,the m RNA levels of the proton-translocating F1F0 ATPase(atp BEFHAGDC)operon were upregulated significantly in the WT-mal S-5’UTR strain compared with those of the WT-p BAD strain.5.The analysis of intracellular ATP measurement revealed that the overexpression of mal S-5’UTR increased intracellular ATP levels by upregulating the m RNA levels of the atp operon.When pho P gene was defective,the intracellular ATP levels of the(35)pho P-mal S-5’UTR strain were higher than those of the WT-mal S-5’UTR strain.6.The WT-mal S-5’UTR-Mgt C::3×Flag,WT-p BAD-Mgt C::3×Flag and(35)pho P-mal S-5’UTR-Mgt C::3×Flag fusion strains were constructed successfully.7.Western-blot analysis showed that the Mgt C level of the WT-mal S-5’UTR strain was lower than that of the WT-p BAD strain.In the mutant pho P gene,the Mgt C level of the(35)pho P-mal S-5’UTR strain was dramatically lower than that of the WT-mal S-5’UTR strain.8.The mal S-5’UTR downregulated the expression levels of pho P and pho Q,thereby leading to lower expression level of mgt C in the WT-mal S-5’UTR strain.The lower m RNA level of mgt C led to the higher expression levels of atp operon,therefore the activity of F1F0-ATPase was enhanced.As a result,the intracellular ATP levels was increased,which led to lower intracellular survival of S.Typhi in macrophage.Conclusions: The mal S-5’UTR downregulates the m RNA level of mgt C in a pho P-dependent manner,which weakens the inhibitory effect of mgt C and results in higher m RNA expression levels of the atp operon that encodes the eight subunits of the proton-translocating F1F0-ATPase.As a result,the intracellular ATP levels may have reached non-physiological levels,resulting in the lower intracellular survival of S.Typhi in macrophage. |
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