ABSTRACT
OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Klebsiella pneumoniae/genetics , Membrane Transport Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites/genetics , Crystallography, X-Ray , Klebsiella pneumoniae/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Multimerization , Structure-Activity RelationshipABSTRACT
Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to remove possible disordered regions based on comparative sequence and structural analysis to salvage the wild-type protein, which failed to crystallize. The 17-residue carboxyl-terminal truncation yielded crystals that diffracted to 3.6â Å resolution. The efflux function measured using minimal inhibitory concentration assays indicated that the truncation decreased, but did not eliminate, the efflux activity of the transporter.
