The Mechanism Of NikR Regulating AhpC Involved In Amoxicillin Resistance Of Helicobacter Pylori

BackgroundHelicobacter pylori(H.pylori)infection is the main cause of inducing gastritis,peptic ulcer and gastric cancer.The elimination of H.pylori is beneficial for the prevention and treatment of these diseases.At present,the resistance of H.pylori to common clinical antibiotics is increasing,and amoxicillin is the first choice for the treatment of H.pylori infection in clinical practice.However,with the widespread use of amoxicillin,the resistance of H.pylori to amoxicillin increased year by year.Understanding the resistance mechanism of H.pylori to amoxicillin is helpful to improve the bactericidal effect of amoxicillin and promote the discovery of new antibacterial drugs.Available studies suggest that mutations in H.pylori penicillin binding proteins(PBPs)are the main mechanism of H.pylori resistance to amoxicillin.Nevertheless,high expression of efflux pumps and altered membrane permeability also mediates the development of amoxicillin resistance in H.pylori.This implies that there are novel mechanisms of H.pylori resistance to amoxicillin and that H.pylori can respond to amoxicillin killing through multiple mechanisms.PurposeTherefore,on the basis of inducing and selecting amoxicillin-resistant strains of H.pylori in vitro,the differentially expressed genes of drug resistant and sensitive strains were identified by transcriptome sequencing,and the new mechanism of amoxicillin-resistance of H.pylori was discussed,providing effective guidance for clinical patient typing,rational use of amoxicillin.Material methods1.In vitro induction and selection of amoxicillin-resistant strains of Helicobacter pylori using an incremental drug concentration method.2.Fluorescence accumulation test was used to determine the accumulation of intracellular fluorescent dye H33342 in different strains.3.The intracellular ROS production of different strains was detected by fluorescence enzyme labeling instrument and flow cytometry.4.The differentially expressed genes were screened by transcriptome sequencing analysis.5.The deletion mutants of pivotal differential genes were constructed by homologous recombination.6.The tolerance of different strains to amoxicillin was compared by agar dilution method,bacterial monoclone formation test and growth inhibition curves.7.The expression of related genes at mRNA level was detected by qRT-PCR.8.The second-generation sequencing technology was devoted to detect and analyze the mutation of related genes.9.The prokaryotic expression protein technology was used to induce expression and purification of target protein.10.EMSA was used for detecting the specific binding of target protein and DNA sequence.11.The kit was detected to the content of intracellular nickel ion in different strains.Results1.Successfully induced AmoRS strains that is amoxicillin resistant strains of Helicobacter pylori in vitro.Multiple mutations were found in pbp1A sequence of Amoxicillin target gene in AmoRS strains,and G595S mutation was drug-resistant.2.Membrane permeability is not involved in amoxicillin tolerance in AmoRS strains.Luciferase assay results showed no difference in intracellular drug accumulation between WT and AmoRS strains.3.The antioxidant capacity of drug-resistant AmoRS strains is enhanced.By comparing the intracellular ROS production of WT and AmoRS strains,the intracellular ROS production of AmoRS strains was significantly lower than that of WT strains.4.Alkyl hydroperoxide reductase AhpC is involved in amoxicillin resistance in Helicobacter pylori.Compared with WT strains,amoxicillin-treated WT group and AmoRS strains with high expression of ahpC.By comparing the tolerance of different strains to amoxicillin,it was found that ΔahpC strains were more sensitive to amoxicillin than WT strains,and AmoRS ΔahpC strains were significantly less tolerant to amoxicillin than AmoRS strains.5.The nickel uptake regulatory protein NikR regulates the expression of AhpC.NikR is known to have two protein conformations,apo and holo,and exists as holo-NikR when nickel ions are sufficient.The presence of NikR binding sites in the ahpC promoter was found by comparing the conserved regions of the promoter sequence;further EMSA studies demonstrated that holo-NikR binds ahpC.qRT-PCR results showed low expression of ahpC in the ΔnikR strain compared to the WT strain.This suggests that the AmoRS strain has a high intracellular nickel ion content,a high presence of holo-NikR and a high expression of AhpC.6.High expression of the nickel transporter protein ExbB results in increased intracellular nickel ion content in the AmoRS strains.Compared with WT strains,the content of Ni ions in AmoRS strains increased.Analysis of nickel ion transporter differential expression genes showed that exbB was highly expressed in AmoRS strains compared with WT strains,but holo-NikR was known to inhibit exbB,suggesting that the inhibitory power of NikR on exbB is reduced in the AmoRS strains.7.The mutation of nikR nucleotide sequence in AmoRS strains results in decreased inhibition of exbB.By detecting nikR gene nucleotide sequence and exbB promoter sequence,it was found that the 3 ’end of nikR DNA sequence was mutated compared with WT strains.The mutated NikR protein was named NikR(M).EMSA assay revealed that the binding of holo-NikR(M)to exbB was reduced,while the binding to ahpC was unchanged.8.ExbB participates in amoxicillin resistance of AmoRS strains by reducing ROS.Due to the high expression of ExbB,the sensitivity of AmoRS ΔexbB strains to amoxicillin was increased by the comparison of AmoRS strains and AmoRSΔexbB strains to amoxicillin.By comparing the intracellular ROS production of AmoRS strains with that of AmoRSΔexbB strains,it was found that the intracellular ROS production of AmoRSΔexbB strains was high,and the qRT-PCR results showed low expression of ahpC,indicating that high expression of ExbB was an important factor in drug resistance.9.ROS can cause NikR to undergo apo-holo allosteric reaction similar to that induced by nickel ions.Because of the decrease of intracellular nickel ion concentration in WT strains,considering that antibiotic treatment can lead to intracellular ROS production,it was found by EMSA that NikR could bind to exbB and ahpC promoters after H2O2 treated.ConclusionsIt was first found that AhpC is involved in amoxicillin resistance in Helicobacter pylori by reducing intracellular ROS,and its expression was regulated by NikR.