The Mechanism Research Of Two Component System ArsRS Involved In The Multidrug Resistance Of Helicobacter Pylori

OBJECTIVEHelicobacter pylori(Hp)is a spiral,micro-aerobic and flagella-like Gram-negative bacteria that can adhere to the surface of human gastric mucosa.It is the only pathogen that has been found in human stomach.H.pylori colonized in the stomach can cause chronic active gastritis,peptic ulcer,even gastric cancer.At present,about half of the world’s population has been infected with H.pylori.Quadruple therapy is the main treatment for H.pylori in clinic,which uses two antibiotics in combination with colloidal bismuth agent and proton pump inhibitor.The most commonly used antibiotics are amoxicillin(β-lactams),clarithromycin(macrolides)and metronidazole(nitroimidazoles).However,in recent years,the resistance of H.pylori to a variety of antibiotics has gradually increased,clinical multi-drug resistant strains have appeared,and the clearance rate of H.pylori has gradually decreased.The two-component system(TCS)is an important signal transduction medium.It is composed of sensor kinases and response regulators.It can sense the stimuli from external conditions and regulate the expression of its own genes to make a stress response.A large number of studies have found that the TCS plays an important role in the multi-drug resistance of bacteria.Three TCSs are expressed in H.pylori,CrdRS,FlgRS and ArsRS.It has not been reported that the TCS of H.pylori is related to its drug resistance.In this study,real-time quantitative PCR was used to detect the expression of the three TCS genes in the wild strains treated with antibiotics.It was found that the expression of ArsRS was reduced,while the expression of the other two TCSs were not changed significantly.Therefore,we operateda series of experiments such as minimum inhibitory concentration,bacterial clone formation,H33342 fluorescence accumulation,mutation rate detection,etc.and further explored whether ArsRS is involved in the multi-drug resistance of H.pylori and its resistance mechanism.METHODS1:Use amoxicillin,clarithromycin,and metronidazole to treat wild strain Hp26695,extract mRNA,reverse transcription to obtain cDNA for qPCR,detect the expression of the three two-component systems,and screen out the TCS that expressing significant differences.2:Construct the gene knockout strain ΔarsS according to the principle of homologous recombination.3:Detect the MIC of wild strain and ΔarsS strain for various antibiotics.4:Measure the growth curve of wild strain and ΔarsS strain.5:Detect the bacterial monoclonals formation of wild strain and ΔarsS strain for a variety of antibiotics.6:Detect the drug efflux ability of wild strain and ΔarsS strain through fluorescence accumulation experiment.7:Detect the base mutation rate and mutation rate change multiples of antibiotic resistance genes in wild strain and ΔarsS strain.8:Treat wild strain separately with amoxicillin,ciprofloxacin,and metronidazole,and detect the expression of all genes in the Hp genome that may be involved in DNA damage repair through transcriptome sequencing technology.mutS and mutY were screened out.9:Detect the expression of mutS and mutY in clinical sensitive strain and clinical multi-drug resistant strain.10:Construct ArsRD52N point mutant strain,and detect the expression of mutS and mutY in ArsRD52N and ΔarsS strain,respectively.11:Construct the gene knockout strains ΔmutS and ΔmutY according to the principle of homologous recombination.12:Detect the MIC of ΔmutS and ΔmutY strains for various antibiotics.13:Measure the growth curves of ΔmutS and ΔmutY strains.14:Detect bacterial monoclonals formation of ΔmutS and ΔmutY strains for a variety of antibiotics.15:Detect base mutation rate and mutation rate change multiples of antibiotic resistance genes in ΔmutS and ΔmutY strains.RESULTS1:The expression of the TCS ArsRS was reduced in Hp26695 after treated with antibiotics,and the expression of CrdRS and FlgRS did not change significantly.2:Based on the wild strain Hp26695,the gene knockout strain ΔarsS was successfully constructed.3:The result of MIC shows that compared with wild strain,ΔarsS strain is less sensitive to multiple antibiotics.4:Comparing the growth curve of wild strain and ΔarsS strain,it was found that knocking out the arsS gene did not affect the growth of bacteria.5:Comparing the bacterial monoclonals formation of wild strain and_ΔarsS strain,it was found that the ΔarsS strain formed more monoclonals on antibiotic plates and enhanced drug resistance.6:In the fluorescence accumulation experiment,compared with wild strain,the accumulation of fluorescent dye H33342 in ΔarsS strain was significantly increased,and its drug efflux ability was not enhanced.7:Comparing the base mutation rate and mutation rate change multiples of the wild strain and the ΔarsS strain,compared with the wild strain,the ΔarsS strain had higher mutation frequency for various antibiotics and the mutation sites of drug resistance genes had also increased.8:Transcriptome sequencing results found that 5 DNA damage repair genes are down-regulated.According to the existing literature reports,only mutS and mutY are involved in bacterial gene mutation.9:mutS and mutY were down-regulated in clinically sensitive strain and clinical multi-drug resistant strain.10:The point mutant ArsRD52N was successfully constructed.Both mutS and mutY were down-regulated in ArsRD52N and ΔarsS strains.11:Based on wild strain,the gene knockout strains ΔmutS and ΔmutY were successfully constructed.12:Compared with the wild strain,the ΔmutS and ΔmutY strains are less sensitive to amoxicillin,metronidazole,and ciprofloxacin,but have no difference in resistance to clarithromycin and tetracycline.13:Measuring the growth curve of the wild strains,ΔmutS and ΔmutY strains were found that knocking out mutS and mutY does not affect the growth ability of the strains.14:Comparing the formation of bacterial monoclonals in wild strain,ΔmutS andΔmutY strains,it is found that the ΔmutS and ΔmutY strains formed more monoclonals on the plates containing amoxicillin,metronidazole,and ciprofloxacin,enhanced resistance.15:Compared with the wild strain,the mutation frequency of the ΔmutS and ΔmutY strains to amoxicillin,metronidazole and ciprofloxacin increased,the mutation sites of drug resistance genes have also increased.CONCLUSIONThe ArsRS TCS plays an important role in multi-drug resistance of H.pylori.By downregulating the expression of the DNA damage repair genes,mutS and mutY,the ability of bacteria to repair mutations is weakened,and gene mutation is increased,thereby the resistance of H.pylori to multiple antibiotics and the ability to adapt to stressful environments is enhanced.