| Objective: In this study,we selected the first gene of several operons(VP1388-1390,VP1393-1399,VP1400-1405 and VP1409-1406)on T6SS1(VP1386-1414)and the operon(VPA1027-1024,VPA1043-1028 and VPA1044-1046)on T6SS2(VPA1024-1046)of V.parahaemolyticus as the target genes.We studied the transcriptional regulation mechanism of CalR on T6 SSs using phenotypic,molecular and biological experiments to provide an understanding of the pathogenic mechanism of V.parahaemolyticus.Methods:1.Bacterial killing assay:WT-p BAD33,Δcal R-p BAD33 and C-Δcal R were successfully constructed of our group peviously.The WT-p BAD33,Δcal R-p BAD33 and C-Δcal R of V.parahaemolyticus strains respectively and E.coli were mixed and cultured in a specific proportion.The bacteria were incubated at 37°C for 4 h.The original bacteria CFU was used as a reference and the bacteria observed after 4 h of for the CFU changes,to determine whether CalR control the bacterial killing ability of V.parahaemolyticus.2.Cell adhesion test:WT-p BAD33,Δcal R-p BAD33 and C-Δcal R respectively were added to the same amount of He La cell precursors and incubated at 37°C,5% CO2 for 90 minutes.The cells were washed three times with PBS,and treated with 0.5% Triton X-100 for10 minutes,centrifuged for 15 minutes and supernatant discarded.Cell cultures were resuspended and serially diluted with DMEM.The cell lysates and input bacteria were plated on LB.The plates were counted for the number of bacteria that adhered to the He La cells to determine whether CalR control the adhesion of V.parahaemolyticus.3.Lac Z fusion:The promoter region of each target gene was cloned into the p HRP309 plasmid between the two endonuclease restriction site and the upstream promoterless lac Z fusion reporter gene.The recombinant plasmids were obtained and transferred into WT and Δcal R,to detremine the β-galactosidase activity.4.Real-time quantitative PCR:The housekeeping gene,16 S r RNA,was used as a reference for the relativequantification of the target genes.Real-time quantitative PCR was used to determine the relative m RNA levels of the WT and Δcal R genes by the ΔCt values by the classicΔCt method.5.Primer extension:The 5’end of the specific primer was labeled with [γ-32P] and the primer was annealed to the complementary region of the m RNA strand to generate c DNA under the action of the reverse transcription AMV enzyme.The same labeled primer was used for sequencing the corresponding DNA fragments.The primer extension products and sequencing materials were concentrated and analyzed in 6.0%polyacrylamide denaturing gel electrophoresis and the results were detected autoradiography.Primer extension experiments identified the transcription initiation site(+1)and the RNase binding site in the-10 to-35 region of the target gene from the sequencing band,and the differences in the transcription of the genes in the wild strain and the defective strain.6.Protein expression and purification:The entire coding region of target gene was amplified using Wild type genomic DNA as template and was cloned between Bam HI and Hind III sites of the plasmid p ET28 a.The recombinant plasmid was transformed by heat shock into E.Coli DH5αcompetent cell for long-term preservation of the recombinant plasmid.The recombinant vector was then knocked into BL21 to express the target protein and screened and verified.The expression of each protein was induced by adding 1 m M IPTG.The expression conditions of different proteins were not the same.The bacteria cultures were centrifuged and the supernatant subjected to Ni-column affinity chromatography to isolate and purify the target protein.7.Electrophoretic mobility shift assay:The DNA promoter region of the gene was amplified by PCR and co-incubated with regulatory protein CalR for 20 min loaded onto a 6% non-denaturing polyacrylamide gel and electrophoresed in 0.5 XTBE buffer for about 90 min at 200 V.The gel was stained with ethidium bromide and examined with a UV transilluminator to detect the binding of CalR to DNA fragments.8.Competitive gel shift assay:Another protein H-NS was added to the system in which the target DNA fragment was co-incubated with the regulatory protein CalR.After finding theoptimum concentration with the negative and positive controls,the concentration of one protein remained unchanged,A gradual increasein the concentration of one protein was done to observe the combination,and vice versa.The reaction was then run with 5.0% polyacrylamide non-denaturing gel electrophoresis toanalyze the competitive relationship between CalR and H-NS.Results:1.CalR activated transcription of VP1388,VP1393,VP1400 and VP1409 directly.1)The results of bacterial killing assay showed that the bacterial killing ability of theΔcal R-p BAD33 was significantly decreased compared with the WT-p BAD33.There was no significant difference between WT-p BAD33 and C-Δcal R,indicating that CalR positively regulates the bacterial killing ability of V.parahaemolyticus.2)The T6SS1 related genes VP1388,VP1393,VP1400 and VP1409 were selected as target genes,and the transcriptional regulation mechanism of CalR was studied by molecular biological experiments.q RT-PCR results showed that CalR could activate the transcription of the above genes.Lac Z reporter fusion assay showed that CalR could activate the promoter region of the above genes.The electrophoretic mobility shift experiments showed that the recombinant protein His-CalR binds to the promoter region of the above gene.2.CalR directly activated the transcription of VPA1027,VPA1043 and VPA1044.1)The result of the He La cell adhesion experiment showed that adhesion rate for the WT-p BAD33 and the C-Δcal R were similar and were significantly higher than theΔcal R-p BAD33,indicating that CalR regulates the adhesion of V.Parahaemolyticus.2)The T6SS2 related genes VPA1027,VPA1043 and VPA1044 were selected as target genes,and the transcriptional regulation mechanism of CalR was studied by molecular biological experiments.Primer extension results showed that the transcription initiation sites of VPA1027,VPA1043 and VPA1044 were located upstream of the coding region,C(-104),T(-120)and C(-36)respectively(the translation initiation site was +1),and their transcriptional activity was activated by CalR.q RT-PCR and Lac Z fusion experiments further demonstrated that CalR does activate transcription of VPA1027,VPA1043 and VPA1044.Electrophoretic mobility shift experiments showed that His-CalR specifically binds to promoter regions of VPA1027,VPA1043 and VPA1044,and His-CalR can antagonize His-H-NS.Conclusions:1.CalR activates the bacterial killing activity of V.parahaemolyticus by regulating the expression of each gene of T6SS1 directly;2.CalR activates the He La cell adhesion activity of V.parahaemolyticus through enhencing on the expression of each gene of T6SS2 directly;3.V.parahaemolyticus CalR act as an antagonist of inhibitory factor H-NS,which activates transcription of gene by competing for H-NS binding sites of the target gene promoter region of T6SS2. |
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