The Roles Of Glutathione In Pseudomonas Aeruginosa Pathogenicity And Antibiotic Resistance

Pseudomonas aeruginosa is the third leading opportunistic pathogen in humans;it can cause serious nosocomial infections.Glutathione(GSH) is one of the most important intra-and extracellular antioxidants,abnormal levels of GSH play an important role in disease pathologies.Evidence shows many pulmonary diseases such as cystic fibrosis(CF) are associated with the lower levels of GSH in the epithelial lining fluid(ELF).GSH is present at high concentrations in normal respiratory ELF, but deficient in CF ELF.Oxidative injury inflicted by the major pathogen, Pseudomonas aeruginosa,in CF lungs is one of the causes in the disease manifestation.Therefore,inhaled GSH therapy has been used in clinical trials to increase the GSH levels in the ELF of CF patients.Studies revealed that the ameliorative effect of GSH against antibiotics-induced damage due to its antioxidant properties;however,GSH has been shown to protect bacterial pathogens against antibiotic treatments,reducing bacterial sensitivity to a number of antibiotics.To understand the conflicting effects of GSH and the relation with P.aeruginosa,we investigated the effect of GSH on the pathogenicity of P.aeruginosa and its sensitivity to different antibiotics.To study the relationship between GSH and the pathogenicity of P.aeruginosa,a promoters library of the virulence factor genes in P.aeruginosa based on the luxCDABE-reporter system was used.The levels of gene expression were monitored by measuring the light production of the luxCDABE reporters.Strains containing the reporters were treated with GSH or buthionine sulfoximine(BSO) and diethylmaleate (DEM) to examine the effect of GSH levels on the expression of virulence factor genes.Glutathione synthetase gene(gshB) mutant PAO1(△gshB) and glutathione reductase gene(gor) mutant PAO1(△gor) were also constructed to confirm the effect of GSH on virulence factor genes.The results indicate that the presence of GSH increased the expression of exoY,exoS,phzA1 and gacA,while no difference was observed with others virulence factor genes tested.PAO1(AgshB) produced markedly less pyocyanin(PCN) than the wild type strain PAOI(P＜0.01);Treating with GSH, the PCN production of PAOI(△gshB) was increased from 0.47μg/mL to 1.73μg/mL (P＜0.01),the PCN production of PAO1 increased from 1.3μg/mL to 1.9μg/mL(P＜0.05),this result indicates that GSH upregulates PCN production. Because exoY,exoS,phzA1 and gacA are related to quorum sensing systems,we investigated the relationship between GSH and QS,and the results indicated that the expression of the gshB and gor gene was downregulated by rhl system.Besides the virulence factor genes,the expression of MexXY-OprM was also decreased in PAOI(AgshB).It was restored to wild type levels when gshB was complemented, confirming the role of gshB.To test the effect of GSH on antibiotic sensitivity of P.aeruginosa,both disk fusion and MIC measurement were used.The results revealed that the presence of 3μg/μL of GSH decreased the sensitivity of P.aeruginosa against kanamycin, erythromycin,cefotaxime sodium,ciprofloxacin,carbenicillin,streptomycin,but interestingly,increased against tetracycline.The MICs against kanamycin was increased from 240μg/mL to 1600μg/mL,the MICs against tetracycline were decreased from 25μg/mL to 1.25μg/mL.The sensitivity to ampicillin and chloramphenicol remained unchanged.The effect of GSH or GSSG on antibiotic activity in PAO1(△gor) mutant was the same as in PAO1.These results indicated that effect of GSH on the sensitivity of P.aeruginosa to different antibiotics differs depending on the types of antibiotics.GSH altered antibiotic sensitivity seems to be unrelated to oxidative stress relief.In summary,this study demonstrates that GSH could increase the expression of exoY,exoS,phzA1,gacA and the production of PCN,and GSH could alter antibiotic sensitivity of P.aeruginosa to different antibiotics.Such effects have to be taken into consideration when GSH treatment is used in pulmonary disearse such as CF where P. aeruginosa infection is involved.These findings highlight the complexity of GSH effects on the host and as well as the pathogens,and uncover new relationship between GSH and the pathogenesis of P.aeruginosa.