Effect Of Helicobacter Pylori Regulatory Protein HP0564 On The Expression Of CagPAI

Objective:The Helicobacter pylori(Hp)virulence factor Cag Pathogenic Island(cag PAI)encodes the type 4 secretory system(T4SS)and the effector protein Cag A.Cag A,the only the oncoprotein in bacteria,enters gastric epithelial cells via T4SS to cause inflammatory responses and release large amounts of inflammatory factors as well as affecting the normal metabolism result in lesions among infected patients.However,under different environmental conditions,cag PAI gene expression varies greatly.Therefore,the investigation for possible mechanisms to regulate cag PAI gene expression could help to understand the pathogenesis of Hp and may offer clinical treatment new ideas.Hp contains 16 transcriptional regulatory proteins,including Rpo D,Rpo N,Fli A,Hrc A,Ars R,HP0222,HP0564,Flg R,Hup,HP1021,Hsp R,Fur,Hsr A,Che Y,Nik R,Crd R.The team of this project conducted a series of studies on the regulatory role and mechanism of these 16 regulatory proteins.The five regulatory proteins were clonally expressed and purified,and then the regulatory proteins bound to the cag A promoter and T4SS promoters were screened by EMSA experiments to further investigate the effect of the transcriptional regulatory proteins on the expression of Cag A and T4SS.Method:1.The construction of regulatory protein library1.1 Construction of the recombinant plasmid"p AKS-IBA7plus-regulatory protein"The hp0564,hup,hsp R,che Y and fli A fragments were obtained by high-fidelity PCR amplification using the NCTC26695 genome as a template.These fragments were ligated to the vector p ASK-IBA7plus and transformed into the BL21Escherichia coli cells to respectively construct recombinant plasmids p AKS-hp0564,p AKS-hup,p AKS-hsp R,p AKS-che Y and p AKS-fli A.1.2 Regulatory proteins expression and purificationBL21 with recombinant plasmids were induced protein expression by tetracycline.these proteins were purified in Strep-Tactin column and verified by SDS-Page and Coomassie Blue Staining.2.HP0564 combines cag A and T4SS promoters2.1 Regulatory protein binding cag A promoter screeningThe cag A promoter fragment was amplified by PCR using primers with Alexa fluorescence group 700 and purified.The promoter fragment respectively incubated for 30 min with the regulatory protein of Strep-HP0564,Strep-Hup,Strep-Che Y,Strep-Hsp R,and Strep-Fli A at five different concentrations:0μM,0.25μM,0.5μM,1μM,and 2μM.Electrophoretic mobility experiments(EMSA)were performed and the blocking strips were observed.2.2 HP0564 binding T4SS promotersThe T4SS main promoter"promoter-cag1","promoter-cag U","promoter-cag X","promoter-Cag L"and"promoter-cag Z"were used as probes for electrophoretic mobility experiments(EMSA)using five different concentrations of"Strep-HP0564"at 0μM,0.25μM,0.5μM,1μM and 2μM,respectively.3.BLAST analysis homologs of the regulatory protein HP0564The amino acid sequence of the regulatory protein HP0564 in NCTC26695 was imported into the NCBI online protein BLAST web(https://blast.ncbi.nlm.nih.gov/).The standard database was selected,and"helicobacter group(taxid:72293)"was excluded from the species,and"PSI-BLAST"was selected for BLAST analysis.4.Construction of NCTC26695 hp0564 mutant strainThe NCTC26695 genome was used as a template,and high-fidelity PCR was performed using relevant primers to obtain the upstream and downstream arm fragments of hp0564,and the kana ~Rfragment was obtained by the same method.These fragments were linked to the linearized plasmid p Bluescript SK II(-)by Clon Express technology and converted into DH5ɑto obtain the recombinant plasmid p Bluescript-hp0564.The recombinant plasmid was electrostatically converted intoNCTC26695 to replace hp0564 gene to kanamycin resistance gene to obtainNCTC26695△hp0564.NCTC26695△hp0564 were validated by PCR and agarose gel electrophoresis using validation primers.5.Effect of HP0564 on cag PAI expression in vitro5.1 Effect of NCTC26695△hp0564 on Cag A expression in vitroBacterial RNA was extracted from liquid cultures of NCTC26695 and NCTC26695Δhp0564 on logarithmic growth periods and then was reversed transcribed into c DNA.The m RNA level expression of cag A was detected using q PCR.Bacterial solution with equal OD was centrifuged and then was boiled for 10min.The protein level expression of Cag A was detected using Western blot.5.1 Effect of NCTC26695△hp0564 on T4SS expression in vitroBacterial RNA was extracted from liquid cultures of NCTC26695 and NCTC26695Δhp0564 on logarithmic growth periods and then was reversed transcribed into c DNA.The first gene of T4SS promoters were detected using q PCR.6.Effect of HP0564 on Cag A expression in Hp-infected AGS cells6.1 Effect of NCTC26695△hp0564 on Cag A expression in Hp-infected AGS cellsAGS cells were cultured in 6-well plates and grew overnight to reach about 80%confluence and divided into third groups:AGS,AGS+NCTC26695 and AGS+ NCTC26695Δhp0564.NCTC26695 and NCTC26695Δhp0564 were pretreated with DMEM/F12 culture medium and added to AGS after 2 hours(MOI=100).These simples were added loading buffer and boiled for 10min.The protein level expression of Cag A was detected using Western blot.6.2 Effect of NCTC26695△hp0564 on T4SS expression in Hp-infected AGS cellsAGS cells were cultured in 6-well plates and grew overnight to reach about 80%confluence and divided into two groups:AGS+NCTC26695 and AGS+ NCTC26695Δhp0564.Total RNA was extracted and used q PCR to detecte T4SS genes expression.Results:1.The construction of regulatory protein library1.1 Construction of the recombinant plasmid"p AKS-IBA7plus-regulatory protein"The result shows that p ASK-hsp R,p ASK-che Y,p ASK-fli A,p ASK-hup and p ASK-hp0564 have been constructed successfully.Strep-HP0564,Strep-Hup,Strep-Che Y,Strep-Hsp R,and Strep-Fli A were induced expression in the BL21containing recombinant plasmids and verified by SDS-page.2.HP0564 combines Cag A and T4SS promoters2.1 Regulatory protein binding Cag A promoter screeningEMSA results indicated that Fli A,Hsp R and Che Y cannot bind directly to the Cag A promoter.Hup showed disappearance of free DNA bands,which suggests that regulatory protein Hup can bind Cag A promoter.The blockade band was seen,and the free DNA fragment was significantly absent,suggesting that HP0564 also can interact with Cag A promoter.2.2 HP0564 binding T4SS promotersThe blocking bands were seen at concentrations of 2μM,1μM,0.5μM and 0.25μM for HP0564,and the free DNA fragments disappeared significantly,which indicated that the T4SS promoters were able to bind to HP0564.3.BLAST analysis homologs of HP0564According to the BLAST results,HP0564 is slightly similar to the hypothetical protein(Campylobacter cuniculorum),Pg NI＿09985(Pyricularia grisea),BHV94＿05910(Clostridiales bacterium)and TPA(Desulfurococcaceae archaeon)in amino acid sequence with E values of 0.5,4.1,8 and 8.7,respectively.The date suggested that HP0564-like protein is not present in the other species.4.Construction of NCTC26695 hp0564 mutant strainPositive clones were screened by Kanamycin BL and verified by colony PCR.The result shows that p Bluescript-hp0564 has been constructed successfully.The strip of NCTC26695△hp0564(2800bp)was larger than the strip of NCTC26695(1400bp),and the strip position was consistent with the strip of"p Bluescript SK II(-)-hp0564" recombinant plasmid.The hp0564 gene of was replaced by kana R to obtain strain NCTC26695△hp0564.5.Effect of HP0564 on cag PAI expression in vitro5.1 Effect of NCTC26695△hp0564 on Cag A expression in vitroThe expression of Cag A in NCTC26695△hp0564 was higher than that of NCTC26695 by q PCR and Western Blot,indicating that the regulatory protein HP0564 can inhibit Cag A expression.5.2 Effect of NCTC26695△hp0564 on T4SS expression in vitroThe results showed that the T4SS gene expression was lower in the NCTC26695△hp0564 group than in the NCTC26695△hp0564 group,except for Cag C,which indicated that the regulatory protein HP0564 can activate the T4SS expression.6.Effect of HP0564 on Cag A expression in Hp-infected AGS cells6.1 Effect of NCTC26695△hp0564 on Cag A expression in Hp-infected AGS cellsWestern Blot results showed that the expression of Cag A and phosphorylated Cag A were increased in the AGS+NCTC26695△hp0564 group compared with the AGS+NCTC26695 group,suggesting that HP0564 inhibits Cag A expression during Hp infection of AGS.6.2 Effect of NCTC26695△hp0564 on T4SS expression in Hp-infected AGS cellsThe results showed that the T4SS gene expression was lower in the AGS+NCTC26695△hp0564 group than in the AGS+NCTC26695 group,except for Cag F,which indicated that the regulatory protein HP0564 can activate the T4SS expression.Conclusion:1.HP0564 can bind cag A and T4SS promoter fragments such as cag1,cag U,cag Z,cag X,cag L.2.HP0564 activates cag M,cag P,cag Q,cag S,cag U,cag V expression and inhibitsCag A protein expression.3.HP0564 activates cag M,cag P,cag Q,cag S,cag U,cag1,cag C,cag V expression and inhibits Cag A protein expression after Hp infection in AGS cells.