Molecular Mechanisms Of SpoT In Biofilm Formation In Response To Oxygen Stress In Helicobacter Pylori

BACKGROUNDHelicobacter pylori infection can cause various diseases such as chronic gastritis and gastric adenocarcinoma.Only by eliminating it can the development of the above diseases be prevented.However,the cure rate has been decreasing year by year in recent years,mainly due to the increasing resistance of Helicobacter pylori.The formation of biofilms by bacteria is one of the important reasons for their resistance to multiple antibiotics.Clinical investigations have found that Helicobacter pylori can generally form biofilms on the surface of gastric mucosa.Studies have shown that a small amount of oxidative stress molecules from the host immune cells can initiate the bacterial stress protection mechanism and stimulate biofilm formation.And Helicobacter pylori infection of gastric epithelial cells can cause inflammation and induce the production of a large number of reactive oxygen molecules(ROS).However,whether ROS molecules released by inflammatory cells can induce Helicobacter pylori to form biofilms has not been reported.(p)ppGpp,also known as magic spot nucleotide,its main function is to regulate the stress response of bacteria and play an important role in adapting to oxygen stress.In Helicobacter pylori,(p)ppGpp is synthesized only by SpoT.It has been found that SpoT plays an important role in the adaptation of bacteria to a variety of stress environments,especially in the process of adapting to oxygen stress,and SpoT is involved in the formation of biofilms.Therefore,SpoT may regulate the formation of H.pylori biofilm induced by oxygen stress.If so,what is the regulatory mechanism?OBJECTIVEThis topic focuses on SpoT,using the WGCNA system to analyze the drug resistance mechanism of biofilm in Helicobacter pylori,that is,SpoT participates in the formation of biofilm under the oxygen stress of Helicobacter pylori by regulating NapA.To further explore the inducible factors of Helicobacter pylori forming biofilm on the surface of gastric mucosa,and whether reactive oxygen molecules can induce the formation of biofilm and its regulation mechanism,so as to provide clues for the treatment of Helicobacter pylori in vivo and the clearance of biofilm.METHODS1.Construction of gene knockout strains ?napA by homologous recombination.2.The colony biofilm method was used to induce the biofilm formation of H.pylori standard and clinical strains.3.Thin layer chromatography was used to detect the accumulation of(p)ppGpp in H.pylori 26695,?spoT,spoT*planktonic state and biofilm(50 ?m hydrogen peroxide induced)state in wild strains.4.Scanning electron microscope(SEM)and laser confocal microscope(confocal)were used to observe the biofilm formation of H.pylori 26695,?spoT,spoT*,?napA in wild strains respectively.5.Agar plate dilution method was used to detect Minimum Inhibitory Concentration(MIC)of wild strains H.pylori 26695,?spoT,spoT*to various antibiotics after biofilm formation.6.Transcriptome detected gene expression of planktonic wild strains H.pylori 26695,?spoT and biofilm wild strains H.pylori 26695,?spoT bacteria(five repeated treatments in each group).WGCNA cluster analysis was conducted to find out the relevant genes that affect biofilm formation and are regulated by spoT.7.Real-time quantitative PCR(RT-PCR)was used to analyze the expression of genes in H.pylori 26695 in planktonic state and H.pylori 26695 in biofilm state.napA gene with the highest expression multiple was screened again from clinical drug resistant strains.8.The expression of neutrophil activating protein NapA in H.pylori 26695 and ?rpoN were determined.RESULTS1.The pre-experimental results showed that low concentration of hydrogen peroxide had no effect on the growth of planktonic bacteria,while high concentration of hydrogen peroxide seriously inhibited the growth of Helicobacter pylori.Therefore,the appropriate concentration range was found to be 50-200 ?M.Considering that the concentration of hydrogen peroxide in the body will not be too high,we chose the final concentration of 50 ?M to simulate the actual situation in the body.2.Compared with planktonic bacteria growing to logarithmic phase,bacteria(p)ppGpp accumulation in biofilm state is more.In addition,compared with H.pylori 26695 and spoT*in wild strains,only a very small amount of(p)ppGpp was accumulated in ?spoT biofilm.3.Comparing the SEM results of wild strain H.pylori 26695 and gene restore strain spoT*,the result of gene knockout strain ?spoT is shown that the extracellular matrix of biofilm decreases and the adhesion degree decreases.Confocal images show that the biofilm thickness decreases in ?spoT,indicating that the ability of strain to form biofilm decreases after knocking out spoT gene,which is consistent with the results of biofilm growth curve.4.Compared with H.pylori 26695 and spoT*in wild strains,the MICs of gene knockout strain ?spoT to various antibiotics after biofilm formation were significantly reduced,indicating that drug resistance decreased after spoT gene knockout.5.Based on the transcriptome data of four groups(standard strains of planktonic bacteria and biofilm,SpoT mutant strains of planktonic bacteria and biofilm)and through comprehensive analysis by WGCNA,gene expression modules that are highly related to biofilm formation and SpoT two indications are obtained.The genes in the modules are subjected to co-expression network analysis by Cytoscape,and 9 key target genes are screened by comprehensive analysis of known gene functions.6.The key target genes were further verified by RT-PCR.The results showed that compared with planktonic bacteria,the expression of 9 key target genes in wild strains H.pylori 26695 after biofilm formation was generally increased.Further screening in clinical drug resistant strains showed that napA may be the key gene.7.napA knockout strain was successfully constructed by using the wild strain H.pylori 26695.8.Compared with the wild strain H.pylori 26695,SEM and confocal results showed that the biofilm formation ability and drug resistance of the strain decreased after knocking out napA gene.9.Comparing the expression of napA in H.pylori 26695 and ?rpoN,the results showed that after knocking out the rpoN gene,the expression of napA was significantly reduced.CONCLUSIONSpoT plays an important role in biofilm formation and multidrug resistance induced by oxygen stress,which may be accomplished by regulating NapA.High expression of NapA promotes the biofilm formation of Helicobacter pylori to reduce oxygen stress damage.