The Preliminary Application Of Comparative Proteomic Analysis On Metronidazole Resistance Of Helicobacter Pylori

Helicobacter pylori(H.pylori) is the main cause of chronic gastritis and peptic ulcer,and is closely related to the occurrence of mucosa-associated lymphoid tissue(MALT) lymphoma and gastric carcinoma.The infection of H.pylori is severely detrimental for human health,thus the treatment and eradication of H.pylori infection is among the most urgent scientific problems.However,there is still no antibiotic that could solely control H.pylori infection,and the eradiation rate of combined antibiotics treatment is also far from satisfying.Recent clinical anti-H.pylori treatments are mostly triple therapies containing two kinds of antibiotics in addition to a proton pump inhibitor(PPI) or colloidal bismuth.The most common antibiotics include Metronidazole(MTZ),Clarithiomycin(CLA),and Amoxicilin(AMX).However,with the prevalent usage of antibiotics,the rate of drug-resistant H.pylori is increasing year by year.Now the reason for unsuccessful H.pylori eradication is generally considered as the emergence of drug-resistance,which is based on discrete mechanisms according to different antibiotics.Metronidazole(MTZ) is a common antibiotic used in triple and quadruple therapy of anti-H.pylori treatment.It is an inactive prodrug requiring for intracellular activation by nitro-reductase to exert its sterilizing effect.Because MTZ have many advantages including high activity,high stability in vivo and low price,it is widely used for treating anaerobic bacteria and protozoa infection,and is the first choice for eradicating H.pylori in clinical. However,the drug-resistance of MTZ is almost the highest among the anti-H.pylori antibiotics now.This phenomenon significantly decreased the effect of MTZ-containing therapies and is regarded as the mainly reason for unsuccessful H.pylori eradiation.The recent explanation about MTZ resistance is as follows:the MTZ-resistance strains of H.pylori could decrease the quantity of nitro-reductase through gene mutation or transcriptional regulation.This change would depress the transformation of MTZ into the sterilizing reduction product,leading to H.pylori's resistance to the antibiotic agent.Most researches presumed that MTZ-resistance might be closely related to the mutations of rdxA, frxA,fdxB,which respectively encode NADPH nitro-reductase,NADPH flavin reductase, oxygen and iron oxygen reduction protein analogues insensitive to oxygen.However,some other researchers found that those genes were equally expressed in partial resistant strains and susceptible strains,which indicated that the formation mechanisms of MTZ-resistance might involve the participation of other genes.This finding demonstrated the necessity of further investigation about other mechanisms underlying H.pylori's resistance to MTZ.The study of metronidazole-resistance mechanisms about H.pylori aims to reveal the mechanisms of bacterial signal transduction after the effect of drugs.However,due to the limitations of the genomics technology,even the mRNA information could not represent the ultimate functions of the protein products.Thus,we have to begin with the executives of life activities-proteins,and monitor their changes in species and quantities after the intervention of drugs.Recently,the most popular technical system includes three steps:separate proteins with two-dimensional gel electrophoresis;identify those proteins with mass spectrometry; store,process,compare and analyze the results of identification using the BIOSIS Previews. Up till now,the sequencing of the entire genome for Helicobacter pylori(H.pylori26695 and H.pyloriJ99) has been completed.Using bioinformatics resources,our research intends to investigate the changes of gene expression pattern in H.pylori after the MTZ-induced transformation from susceptible strain to resistant strain through the high-throughput proteomics technology,thereby provide meaningful clues for further illustrating the mechanisms of H.pylori drug-resistance.Holding that purpose,we did the following work:1.Induction and identification of MTZ-resistant strain in vitroH.pylori26695 susceptible strain was cultured in vitro,and the H.pylori26695 MTZ resistant strain was constructed by continuously doubling MTZ concentration.Minimal inhibitory concentrations(MICs) of MTZ to H.pylori 26695 were evaluated before and after MTZ induction.MIC increasing more than 32 times(including 32 times) was used as the criteria for successful induction.In order to assess the stability drug resistance,isolated MTZ-resistant strain was transferred 5 times before the performance of reverse assay and the corresponding evaluation of MIC.The rdxA was then amplified by PCR and sequenced for mutation.We successfully induced a stable H.pylori26695 MTZ-resistant strain,whose tolerance to MTZ was 128 times more than that of the susceptible strain.The MIC kept being the same as before the reverse assay,and the rdxA sequencing results presented insertions at 3 sites.This work provided an experimental model for analyzing the mechanisms of MTZ-resistance in H.pylori.2.Construction of the two-dimensional gel electrophoresis map about Helicobacter pylori susceptible strain and MTZ-resistant strainThe susceptible and resistance strain were harvested by centrifugation.The bacterial proteins were extracted and separated by 2-dimentional electrophoresis.The proteins were stained by silver nitrate.The protein spots in the gels were scanned by ImageScanner and analyzed by 2D Elite software to identify the differential proteins.We found that in the MTZ- resistant strain 55 spots showed apparently changed on expression levels.3.Mass spectrometry of the differential proteins about Helicobacter pylori susceptible strain and resistant strainThose differential spots were excised and identified by MALDI-TOF-MS proteomic system(Applied Biosystems) and information about the differential proteins was obtained. The data was analyzed by protein databases(http://www.matrixscience.com) and(http: //www.expasy.ch/tools/peptident.html) on the internet and 24 proteins were identified and respectively belonged to 16 different protein categories.Those proteins were involved in various cellular functions including cell growth,stress response,pathogenesis,Synthesis, metabolism,replication and translation.Expression levels of some proteins such as ureG, hypB,dnaK,groEL and ftsZ changed significantly.