The Influence Of Iron-Deficiency On LuxS Expression In Helicobacter Pylori

ObjectiveHelicobacter pylori(H.pylori)is an important factor causing gastrointestinal diseases and related cancers.Cag pathogenic islands and other virulence genes play a key role in the pathogenesis of H.pylori.The study of the regulation mechanism of virulence gene expression is crucial.Iron deficiency will increase the risk of gastritis and gastric cancer,and increase the colonization of H.pylori in mice,Cag A expression levels and inflammatory capacity,indicating that environmental iron concentration regulates H.pylori toxicity.Colony induction is a mechanism by which bacteria sense population concentrations and coordinate population behavior,particularly virulence gene expression.Auto Inducer-2(hereinafter abbreviated as AI-2)is a quorum-sensing signal molecule that is widely present in various types of bacteria.Lux S is a key protein involved in its synthesis,but there is currently limited research in H.pylori.Studies have found that AI-2 is associated with the biofilm formation and inflammation of Helicobacter pylori.Therefore,studies on the expression of Lux S and the synthesis of AI-2 are helpful for understanding the pathogenic mechanism of H.pylori.This study focuses on elucidating the relationship between the synthesis of Lux S and AI-2 in H.pylori and the effect of environmental iron concentration on the expression of Lux S and the synthesis of AI-2.Method1.Construction of H.pylori NCTC26695 ?lux SThe complete genome sequence of H.pylori NCTC26695(hereinafter abbreviated as NCTC26695)was obtained from NCBI,and the primers of the upstream and downstream gene arms were designed to carry corresponding restriction sites,and the upstream and downstream arm genes and resistance to kanamycin were obtained.Genes(Kana R)were constructed into pLB vectors for screening and validation.The correctly constructed fragment of the p LB plasmid was recut and the upstream/downstream arm of lux S was grafted into the p Bluescript SK II(-)vector.The correct recombinant p Bluescript SK II(-)plasmid was successfully screened and verified.According to the principle of homologous recombination,it was transformed into the NCTC26695 strain by electroporation transformation method,and the lux S gene mutant was screened out by culture and passaged,and the genome sequencing and PCR were performed.Identification,lux S gene mutant(hereinafter referred to as NCTC26695 ? lux S),to provide a control for follow-up experiments.2.Preparation and verification of anti-Lux S antibodyThe lux S gene primer was designed from the NCTC26695 genome sequence and the extracted nc UT26695 gene was used as a template to construct the lux S gene and then inserted into the p LB vector for screening and validation.The lux S gene was then ligated into the p ASK-IBA3 C vector and transformed into E.coli BL21 and used.Tetracycline induces expression and purification of the protein is performed using a Strep tag purification column and the purity and concentration of the protein are verified.The purified Strep-Lux S protein was diluted to a specific concentration and mixed with the corresponding Freund's adjuvant.The New Zealand white rabbits were immunized subcutaneously and immunized four times for two weeks.After the completion of immunization,blood was taken on the third day to prepare serum and antiLux S polyclonal serum was obtained.The specificity of the protein extracted by NCTC26695 and NCTC26695 ?lux S was verified by Western Blot.3.Compare the impact of iron deficiency on Lux S at protein level3.Effect of iron deficiency on transcription and protein expression of lux S in NCTC26695The NCTC26695 was cultured in liquid and divided equally into two groups.The treatment group was added with 2,2'-dipyridyl(ie divalent iron ion chelating agent,hereinafter referred to as Dip)to a final concentration of 200 u M in the culture broth,and the untreated control group.The bacteria were collected at 2h,4h and 8h after culture,RNA was extracted,RT-PCR was performed with random primers to reverse the c DNA,and bacterial 16 s RNA was used as the internal reference and corresponding internal lux S q PCR primers were designed for real-time fluorescence quantitative PCR.Differences in expression levels of lux S gene transcription between groups.Similarly,the bacterial proteins collected at 4h and 8h after the same culture were treated with the above-prepared anti-Lux S polyclonal primary antibody were subjected to Western Blot to confirm the difference in Lux S expression at the protein level.4.The effect of iron deficiency on AI-2 production per concentration of NCTC26695:The liquid cultured NCTC26695 was divided equally into two groups.The treatment group was supplemented with Dip in the culture medium to a final concentration of 200 u M,and was collected at 4 h,6 h,10 h,and 20 h after incubation with the untreated control group NCTC26695 and the same bacterial concentration of NCTC26695 ?lux S.Bacterial culture fluid was centrifuged to take supernatant.The concentration of AI-2 in the sample was indirectly measured by the Autoinducer Bioassay method.NCTC26695 ?lux S was used as the background value to balance the effects of factors other than Lux S on AI-2 in NCTC26695.AI-2 Concentration/Bacterial Concentration" calculates the AI-2 content produced per unit concentration of NCTC26695.5.Statistical analysisSPSS22.0 and excel 2016 were used for data collation,statistical analysis and mapping,and IPP 6.0 was used for WB grayscale analysis.Results1.Obtaining Helicobacter pylori NCTC26695 ?lux SPositive transformants successfully transformed by electroporation were further screened by colony-PCR,and the screened colonies were subjected to multiple passages to extract the genome for PCR and sequencing.The NCTC26695 lux S mutant strain NCTC26695 ?lux S was obtained.2.Obtain anti-Lux S polyclonal antibody serumUsing Western-Blot method,using successfully prepared serum as the primary antibody,after dilution 1:1000,the NCTC26695 and NCTC26695 ?lux S bacterial proteins on PVDF membrane after SDS-PAGE and electroporation were incubated at 4°C overnight and further incubated.The anti-Lux S polyclonal antibody serum was obtained by negative control validation against NCTC26695 lanes with a size of approximately 19 k D in the positive and negative bands and using the corresponding size position of the NCTC26695 ?lux S lane.3.Iron deficiency environment promotes transcription and expression of H.pylori NCTC26695 lux S geneAfter real-time PCR,the SPSS analysis showed that the m RNA levels of lux S in the Dip group were higher than those in the control group at 2hours(short hand 2h),4h,and 8h after Dip treatment(P<0.05).Among them,the difference in the 2h group was the largest,and the difference between the 4h and 8h was relatively reduced.Western-Blot results were calculated by IPP gray scale and analyzed by SPSS.After 4 and 8 hours of Dip treatment,Lux S gray value of the treatment group was higher than that of the control group,and the difference was statistically significant(P<0.05).The 8h difference is greater than 4h.4.Iron deficiency environmental promotion unit concentration NCTC26695 AI-2production:At 2h,4h,6h,10 h and 20 h after Dip treatment,the mean AI-2 production per unit of H.pylori in the treatment group was higher than that in the control group,and the difference between the treatment group and the control group at 4h,6h,10 h,and 20 h was statistically significant.Significance(P<0.05).The difference between 6h and 10 h reached the maximum.Conclusion:1.Under conditions of in vitro culture,the iron-deficiency environment promotes the transcription and expression of the lux S gene of NCTC26695.2.In vitro culture conditions,iron deficiency environment promotes the secretion of AI-2 in the unit NCTC26695.3.Helicobacter pylori lux S is a key gene that affects the synthesis of AI-2.