SpoT Is Involved In The Formation Of Biofilm And Multidrug Resistance Of Helicobacter Pylori By Regulating GluP

OBJECTIVEHelicobacter pylori(H.pylori)is a spiral,microaerophilic gram-negative pathogen,and is the only known pathogen colonized in the human stomach,and its long-term colonization of human gastric mucosal epithelial cells and mucus layers,is the main cause of chronic gastritis and peptic ulcer disease,and it also leads to gastric cancer and mucosa-associated lymphoid tissue lymphoma(MALT),as the important risk factors.The rate of H.pylori infection is high,at present,the number of infected people in the world is about more than half.Therefore,the treatment of Helicobacter pylori eradication and infection is the key measure to prevent and control many gastric diseases.First-line therapy for the treatment of H.pylori clinically mainly triple,quadruple therapy,which requires the use of antibiotics.At present,the types and numbers of antibiotics that can be used for the treatment of H.pylori are not many.Antibiotics commonly used are amoxicillin or metronidazole and clarithromycin.However,the resistance of H.pylori to multiple antibiotics is more and more serious,which is also an important reason for the failure of treatment of H.pylori infection.Because of its high infection rate and difficult to cure characteristics,so the antibiotic resistance of H.pylori has been our research focus.There are many possible mechanisms of H.pylori drug resistance,such as the generation of inactivated enzymes,the change of the target of drug action and the barrier of antibacterial penetration.The formation of biofilm is an important cause of H.pylori drug resistance.At the same time,a large number of studies have shown that H.pylori can form biofilms both in vivo and in vitro.(p)ppGpp is a signaling molecule that regulates bacterial rigorous response.Rigorous response,is the occurrence of changes in gene expression patterns,after external environment pressure,thus affecting the bacterial viability and stress-related gene expression.A large number of studies have shown that(p)ppGpp is closely related to the formation of bacterial biofilm and drug resistance.The accumulation of(p)ppGpp in H.pylori is related to biofilm formation and multi-drug resistance.spoT is an enzyme that regulates(p)ppGpp.In E.coli,the enzymes that regulate(p)ppGpp are both relA and spoT,whereas there is only one spoT in H.pylori.Therefore,we investigated whether spoT in H.pylori correlates with biofilm formation andmultidrug resistance.The efflux pump system is composed of a series of transport proteins,also known as multi-drug transporters,plays a role in the discharge of toxic substances from bacteria,thus protecting the bacteria from environmental stress.Therefore,the most basic mechanism of drug resistance is the efflux pump system to excrete the drug out of the bacterial cells.It has been proved that the spoT regulated proton pump involved in H.pylori resistance.Meanwhile,it has been reported that the expression of efflux pump genes is related to the formation of biofilm and drug resistance in other bacterial.The expression of gluP was significant different between planktonic bacteria and biofilm bacteria,which was identified by qPCR and further studied using variable scientific methods.This study aims to study whether spoT was involved in the formation of H.pylori biofilm and its multi-drug resistance through regulating the expression of efflux pump gene gluP.This provides a new potential drug target for solving the multi-drug resistant problem of H.pylori and effectively guides the rational use of antibiotics in clinical practice.METHODS1.MIC and growth inhibition curves of wild-type H.pylori 26695 and ?spoT for multiple antibiotics were determined.2.In the(p)ppGpp accumulation test,it was found that after the biofilm formation,the expression level of(p)ppGpp increased;3.The planktonic and biofilm-forming wild-type H.pylori 26695 and ?spoT were used to photograph SEM,confocal,respectively.And biofilm thicknesses were determined.4.The MICs of biofilm-forming wild-type H.pylori 26695 and ?spoT were determined for the various kinds of antibiotics.5.H33342 accumulation assays were performed to compare the difference in efflux ability between biofilm-forming wild-type H.pylori 26695 and ?spoT.6.The total mRNA of chloramphenicol resistant strains and biofilm-forming wild-type H.pylori 26695 and ?spoT was extracted,and was then reversely transcribed into cDNA.Transporters genes expressed differently were then detected by qPCR.7.After treated by different antibiotics for different time and concentration,the expression of efflux pump gene gluP in wild-type H.pylori 26695 and dspoT,was detected by qPCR.8.Gene knockout mutant strain ?gluP and ?gluP-MDR was constructed using wild-type H.pylori 26695 and MDR as parent strain,respectively.9.The planktonic and biofilm-forming wild-type H.pylori 26695 and ?gluP were used to photograph SEM,confocal,respectively.And biofilm thicknesses were determined.10.MIC and growth inhibition curves of ?gluP for multiple antibiotics were determined.11.The MICs of biofilm-forming ?gluP were determined for the various kinds of antibiotics.12.H33342 accumulation assays were performed to compare the difference in effluxability between wild-type H.pyLlori 26695 and ?gluP,biofilm-forming wild-type H.pylori 26695 and ?gluP,respectively.13.The MICs of MDR and ?gluP-MDR were determined for the various kinds of antibiotics.14.The expression of transporter GluP in clinical multi-resistant strains and clinical sensitive strains were determined.15.The expression of transporter GluP in wild-type H.pylori 26695 and ?rpon were determined.16.Observed morphology of ?gluP biofilms by SEM,when the D-glucose concentration in the medium was 1.00 g/L and 4.00 g/L.RESULTS1.The MICs,growth inhibition curves,confocal microscopy,and MICs of biofilm-forming strains showed that the susceptibility of ?spoT was higher than that of wild-type H.pylori 26695.At the same time,the formation of biofilms decreased the susceptibility of both strains,but susceptibility of biofilm-forming wild-type H.pylori 26695 is still lower than that of biofilm-forming ?spoT.2.The results of SEM and confocal showed that the ability of forming biofilm in?spoT was lower than that of wild-type H.pylori 26695.The determination of thickness of biofilm in these two strains also showed that.3.H33342 accumulated more in biofilm-forming strains than in planktonic strains.And it accumulated more in ?spoT than in wild-type H.pylori 26695.4.The expression of transporter GluP was higher in biofilm-forming strains than in planktonic strains,and it was higher in wild-type H.pylori 26695 than in ?spoT.After respectively treated with different kinds of antibiotics for different time and concentration,the expression of transporter GluP was higher in wild-type H.pylori 26695 than in ?spoT.At the same time,transporter GluP showed a higher expression in MDR.5.Gene knockout mutant strain ?gluP and ?gluP-MDR were constructed successfully using wild-type H.pylori 26695 and MDR,respectively.6.The growth curve of ?gluP showed little difference compared with that of wild-type H.pylori 26695.7.The MICs,growth inhibition curves,confocal microscopy,and MICs of biofilm-forming strains showed that the susceptibility of ?gluP was higher than that of wild-type H.pylori 26695,except clarithromycin.8.H33342 accumulated more in ?gluP than in wild-type H.pylori 26695.And it also accumulated more in biofilm-forming ?gluP than in biofilm-forming wild-type H.pylori 26695.9.The MICs of MDR strain and ?gluP-MDR showed that the susceptibility of MDR was higher than wild-type H.pylori 26695,and after knocking out gluP,the susceptibility was increased.10.The expression of transporter GluP in clinical multi-resistant strains was higher than that in clinical sensitive strains.11.The expression of transporter GluP in wild-type H.pylori 26695 was higher than that in ?rpon.12.After knockout of gluP,when the concentration of D-glucose was 1.00g/L,the ability of forming biofilm was higher than that was 4.00g/L.CONCLUSIONspoT plays an important role in the formation of H.pylori biofilm and multidrug resistance,which can enhance the ability of H.pylori to form biofilm by regulating the expression of efflux pump gene gluP,and promote the ability of H.pylori to efflux a variety of antibiotics,reduce the damage of H.pylori by antibiotics,make H.pylori better to adapt to the antibiotic stress environment,enhance the ability of H.pylori to tolerance multiple antibiotics.