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Title: Effect of the global response regulator MorA on the multi-drug efflux pump MexCD-OprJ IN Pseudomonas aeruginosa
Keywords: global regulator, MorA, multi-drug efflux pump, MexCD-OprJ, MexAB-OprM, Pseudomonas aeruginosa
Issue Date: 19-Aug-2010
Citation: SANDHYA VARADARAJAN (2010-08-19). Effect of the global response regulator MorA on the multi-drug efflux pump MexCD-OprJ IN Pseudomonas aeruginosa. ScholarBank@NUS Repository.
Abstract: SUMMARY P. aeruginosa is an opportunistic human pathogen having intrinsic resistance and is often capable of developing acquired multidrug resistance during antibiotic therapy. Among the major families of bacterial multidrug efflux transporters, clinically relevant antimicrobials are primarily accommodated by the RND family. Of all the RND pumps in P. aeruginosa, only MexAB-OprM and MexXY-OprM (constitutive pumps) and MexCD-OprJ and MexEF-OprN (inducible pumps) have been reported to provide significant resistance to antibiotics when stably overproduced upon mutations. We have previously identified a sensory regulator MorA that coordinately controls motility and biofilm formation in P. aeruginosa and is predicted to be involved in c-di-GMP signaling by virtue of its GGDEF and EAL domains. A global gene expression profiling demonstrated that over 80 genes were affected by the loss of morA in P. aeruginosa PAO1 and the most significantly affected ones were that of the mexCD-oprJ pump. Furthermore, the effect on the pump MexCD-OprJ by MorA was occurring in an nfxB-independent manner. Interestingly nfxB (the only characterized negative regulator of mexCD-oprJ), operon mexCD-oprJ and morA are all located adjacent to each other on the P. aeruginosa PAO1 genome. Our aim was to study the effect of the response regulator MorA on the inducible pump MexCD-OprJ in P. aeruginosa. Promoter mexCD-oprJ activity, RNA levels of the pump and the antibiotic resistance phenotype were studied in wild type and morA deletion strains of K767 and K1119 (mexAB-oprM deletion) P. aeruginosa. Promoter mexCD activity increased in a time dependent manner in the absence of MorA in both K767 and K1119, indicating that MorA has a negative effect on the promoter activity. However, transactivaton with morA in the mutants did not restore promoter activity to wild-type levels indicating that MorA had an indirect effect on the pump. There was a marginal but reproducible time dependent fold increase in the RNA levels of mexC in K1119 morA deletion strain compared to K1119. However, there was no increase in the RNA levels of the pump for K767 morA deletion strain. Both promoter and RNA fold increase of MexCD-OprJ pump in morA mutants compared to wild type was higher in K1119 background and not K767. From gene expression analysis, we could conclude that MorA has a marginal, time and MexAB-OprM dependent effect on the mexCD-oprJ expression. Antibiotic phenotype assay indicated that K767 had a significant two fold higher MIC than the morA deletion strain for antibiotics known to be specific to MexCD-OprJ.However, there was no change in MIC values between K1119 and its morA mutant. Moreover, complementation of phenotype was observed when morA was provided in trans in the K767 morA deletion strain confirming the direct role of MorA in affecting the resistance phenotype. From the antibiotic susceptibility assay we could conclude that MorA has a MexAB-OprM dependent positive effect on the drug resistance phenotype of MexCD-OprJ. Since the gene expression levels of the pump does not explain the phenotype observed, we concluded that MorA affects MexCD-OprJ in a posttranscriptional manner. This may involve signaling via the secondary messenger c-di-GMP. The fact that an inducible pump is being affected by a biofilm and motility regulator, in a constitutive pump dependent manner in P. aeruginosa is a novel finding from this study.
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