The Role Of OxyR In The Process Of Bacterial Resistance To Fluoroquinolone Antibiotics

Research Background:Bacterial resistance is a main cause for the failure of treatment on clinical infections,whicheven result in the loss of life for patients.Recent studies have shown that fluoroquinolone antibiotics may produce reactive oxygen species（ROS）and participate in bacteriocidal process.When bacteria are treated with sublethal concentrations of drugs,the frequencies of bacterial mutations increase,which may promote the generation of drug resistant strains.Oxy R is a transcription factor which activity is regulated by sulfhydryl/disulfide bond conversion.It can sense changes in redoxenvironment in bacterial cells,participate in bacterial anti-oxidation,inhibition of spontaneous mutations,pathogenicity,iron homeostasis,and outer membrane proteins metabolism.Oxy R participates in the regulation of ROS levels in bacteria,but its role in antibiotic resistance is still unclear.Research purposes:The aims of this study are to explore the role of oxy R in bacterial drug resistance.In particular,we want to clarify the regulation of E.coli cellular redox environment by Oxy R and correlationbetween occurrence of the mutation and the redox environment when bacteria are exposed underoxidative stress.Furthermore,we want to understand the mechanism in detail about Oxy R-mediated cellular events in the generation of bacterial drug resistance,in order to provide new thoughts forovercoming antibiotic resistance.Research methods and results:1.Construction of mutant strains of Escherichia coli that regulate gene defects related to bacterial redox microenvironmentTo knochout the genes related to redox regulation in E.coli,plasmid p KD4 was used as a template to amplify homologous targeting fragments with kanamycin-resistant fragments firstly.Then the DNA fragment was electrotransfered into E.coli MG1655 containing the helper plasmid p KD46 expressing Red recombinase.After performing homologous recombination to replace the redox-related target genes with resistant fragments,the positive transformants were screened with kanamycin resistance plates.Finally plasmid p CP20 expressing FLP recombinase was transferred into the selected clones to delete the resistance genes.PCR and DNA sequencing technology were used to identify the gene knockout results.In this study E.coli MG1655 oxy R^-,ahp C^-,kat G^-,grx A^-,grx B^-,grx C^-,gsh B^-mutant strains were prepared..2.Induction of norfloxacin resistant Escherichia coli strainsDrug resistant E.coli strains were developed by challenged with increasing sublethalconcentration of norfloxacin in the culture medium.Induced from wild type E.coli with MIC 0.078μg/m L aganist norfloxacin,a drug resistant strain R-Norf-E.coli-WT was obtained with maximum tolence concentation of norfloxacin.MIC for R-Norf-E.coli-WT against norfloxacin was 31.25μg/m L.In contrast,induced from oxy R^-E.coli mutant with MIC 0.078μg/m L aganist norfloxacin,a drug resistant strain R-Norf-E.coli-oxy R with maximum MIC of 625μg/m L was obtained.Thedeficiency of Oxy R caused R-Norf-E.coli-oxy R could tolerant nearly 20 times higher concentration of norfloxacin compared to strain R-Norf-E.coli-WT.This result indicates that oxy R is a key anti-drug resistance gene in the process of E.coli resistance to norfloxacin.Beside norfloxacin,thesensitivities of these two resistant strains towards the other two fluoroquinolone antibiotics ofloxacin and levofloxacin were also largely reduced,while the sensitivities of the drug resistant strainsagainst the antibiotics with other mechanism e.g.kanamycin and gentamycoin were not dramatically changed..3.Roles of redox-related genes on the mutation rate of bacteriaTo clarify the mechanism of oxy R and other redox microenvironmental regulation-related genes in the process of drug resistance,we investigated the influence of these genes on the mutation rate of bacteria.After the treatment with sublethal bactericidal antibiotic norfloxacin and gradient dilution,E.coli strains were inoculated in blank plates and rifampicin-resistant plates.Subsequently thebacteria were cultured overnight,and the clones were counted.Then,the MSS maximum likelihood method was used to estimate the number of mutations（m）in each culture,and the mutation rate（μ）was calculated.The results showed that the mutation rate of the norfloxacin treated bacteria wassignificantly higher than that of the untreated group.In the strains lacking the oxy R gene,the mutation rate was significantly higher than that of the wild type before and after norfloxacin treatment,indicating that when the bacteria lacking oxy R gene meight be more susceptible to oxidative stress,resulting in more severe DNA damage,leading to an increase in mutation rate.4.Oxy R Regulates Oxidative Stress Caused by Hydrogen PeroxideOxy R is a redox regulator that senses peroxide,which can sense the elevation of H₂O₂ level and induce the response of the antioxidant system.To investigate the changes of the genes regulated by Oxy R under oxidative stress,we treated E.coli WT and mutant strains with H₂O₂ and detected the expression levels of redox-related genes by RT-q PCR.With increase of H₂O₂ concentration,them RNA levels of ahp C,ahp F,kat G,trx B,trx C,and grx A genes in wild-type strains increased.Them RNA level of kat G got the highest enhancement,indicating that kat G plays a very important role in eliminating H₂O₂.In the oxy R gene knockout strain with,the m RNA levels of ahp C,ahp F,grx A,trx B,trx C,especially kat G genes were significantly down-regulated under oxidative stress.Many genes in Trx amd Grx systems including trx B,trx C,and grx A are regulated by Oxy R.On the other hand the activity of Oxy R is mediated by the Trx1 and Grx1,displaying a typical negative feedback effect.5.Transcriptome analysis of E.coli wild-type and drug-resistant bacteriaTo understand the mechanism of Oxy R in regulating E.coli’s reprogramming process against norfloxacin,we used RNA-seq technology to analyze WT,E.coli-oxy R^-,R-Norf-E.coli-WT and R-Norf.-E.coli-oxy R^-simultaneous transcriptome analysis.The results showed that compared with R-Norf-E.coli-WT,the number of differentially expressed genes in R-Norf-E.coli-oxy R^-increasedtwice,indicating that the loss of Oxy R makes more genes involved in bacterial reprogramming topromote higher levels of resistance.Among them,the expression of genes involved in iron transport,iron efflux and Fe-S cluster assembly system and other iron metabolism were significantly up-regulated in R-Norf-E.coli-oxy R^-,but the iron content in the bacteria was significantly reduced.In addition,the level of ROS in R-Norf-E.coli-oxy R^-is significantly increased,indicating thatoxidative stress and iron homestasis in bacteria participate in bacterial drug resistance in concert.6.Detection of total reducing activity of bacterial thioredoxin and glutaredoxinTRFS-green is a fluorescent probe used to detect mammalian thioredoxin reductase（Trx R）.In in vitro experiments,TRFS-green is a selective substrate for bacterial Trx and Grx.This allowsTRFS-green to be used as a specific probe to detect the overall activity of Trxs and Grxs in bacterial cells.The role of different Trx and Grx in maintaining the redox environment in the cell was studied.It was found that E.coli Grx2 and Grx3 showed higher activity than other proteins in reducingdisulfide bonds of small molecules.In addition,the probe was used to detect the reducing ability of E.coli upon the treatment with antibiotics at MBC concentration.The results showed that thebacterial disulfide bond reductase activities were affected during the bacterial sterilization process.TRFS-green can be used as a powerful tool to detect the redox regulation of bacteria.E.coli Grx2and Grx3 were shown to be the key factors in maintaining the redox environment in bacterial cells.Using this probe,we detected the overall reduction ability of Trx and Grx in strains such as WT,E.coli-oxy R^-,R-Norf-E.coli-WT and R-Norf-E.coli-oxy R^-,and the activity of Trx and Grx in R-Norf-E.coli-WT and R-Norf-E.coli-oxy R^-resistant bacteria was significantly lower than that of sensitive strains.This result was agrreable with the observation that level of ROS in resistant bacteria wassignificantly higher than that in sensitive bacteria.This indicate that cellular overall reducting ability of Trx and Grx activity may contribute to bacterial resistance.In summary,upon the oxidative stress,Oxy R can quickly sense the presence of reactive oxygen species,regulate the expression of downstream antioxidant genes to keep redox balance.We havefound that Oxy R regulated redox environment is correlated with bacterial drug resistance.E.coli-oxy R^-mutant is more likely to develop high-level drug resistance upon the treatment withnorfloxacin.The mutation rate of E.coli-oxy R^-is significantly increased compared with wild type E.coli.These results indicate that oxy R is a key anti-resistance gene.Furthermore,we have found that in the drug resistant strain R-Norf-E.coli-oxy R^-,the genes involving iron metabolism aresignificantly up-regulated,but cellular iron level is decreased.In addition,the level of ROS in the bacteria is significantly increased and the Trx and Grx overall activities are reduced.These results strongly suggest that the coordinated regulation between bacterial redox environment and ironmetabolism is closely linked to bacterial drug resistance.