The pharmacological properties of wild-type MexB

The pharmacological properties of wild-type MexB PF-6463922 and the mutant were compared in detail with cytotoxicity assays and the measurement of drug transport. To study the effect of the FAFA mutation on the ability of MexB to confer resistance to cells against antibiotics, a plasmid encoding the MexAB-OprM operon containing wild-type MexB or FAFA MexB was expressed in E. coli BW25113 cells lacking the MexAB homologues AcrAB (BW25113 ΔAcrAB). MexAB-OprM expressed in E. coli displays the same substrate specificity and properties as in P. aeruginosa (Srikumar et al., 1998; Krishnamoorthy et al., 2008; Welch et al., 2010). Using E. coli as host has obvious advantages in comparison

with using P. aeruginosa, such as nonpathogenicity. Additionally, the thick mucoid layer contributes to intrinsic resistance in P. aeruginosa, making it difficult to do mechanistic work relating to the expression of the MexAB-OprM efflux pump with a range of different drugs. Wild-type MexB and the FAFA mutants were expressed at a similar level in the cytoplasmic membrane of the E. coli cells (Fig. 2a). The FAFA mutation impedes the ability of MexAB-OprM to confer resistance to antibiotics that act inside the cell (Table 1), such

as the coumermycin antibiotic novobiocin (DNA topoisomerase inhibitor); norfloxacin, nalidixic acid, ciprofloxacin and mitoxantrone (DNA topoisomerase inhibitors); erythromycin and minocycline (protein synthesis inhibitors) and the DNA intercalaters doxorubicin, ethidium and Rhodamine 6G. Wild-type MexB were able to give up to > 32-fold resistance against

these antibiotics, while the MIC values for the FAFA mutant are either not different Tau-protein kinase from that of the non-MexB-expressing control cells or significantly lower than that of wild-type MexB (Table 1). In contrast, the FAFA mutation had no effect on resistance against toxic compounds that act on the membrane, such as the detergents sodium dodecyl sulphate (SDS) and DDM or the membrane probes 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) and tetraphenylphosphonium (TPP). For these compounds, the cells expressing mutant and wild-type proteins displayed similar MIC values which were significantly higher than that of the nonexpressing control cells (Table 1). We also prepared and tested the effect of the individual Phe to Ala mutations on drug efflux. The F5A mutant plays a more significant role in the phenotype; however, for some drugs, the full effect is only observed in the presence of both mutations (Table 1). Owing to the high intrinsic resistance of the MexAB-OprM expression vector to β-lactam antibiotics, the effect of β-lactams on the activity of wild-type and FAFA MexB were tested by cloning of MexB and FAFA MexB into a pET 41a(+) plasmid. The plasmids were propagated in E. coli BW25113 ΔAcrB cells.

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