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Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles.

ABSTRACT: Recent mutations in RND efflux pumps in clinical strains have further increased multidrug resistance. We show that R717L and R717Q substitutions (found in azithromycin-resistant Salmonella enterica spp.) in the Escherichia coli efflux pump AcrB dramatically increase macrolide, as well as fluoroquinolone, resistance. On the other hand, cells became more susceptible to novobiocin and β-lactam cloxacillin. We urge the control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

SUBMITTER: Zwama M

PROVIDER: S-EPMC9017336 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles.

Zwama Martijn M Nishino Kunihiko K

Antimicrobial agents and chemotherapy 20220321 4

Recent mutations in RND efflux pumps in clinical strains have further increased multidrug resistance. We show that R717L and R717Q substitutions (found in azithromycin-resistant Salmonella enterica spp.) in the Escherichia coli efflux pump AcrB dramatically increase macrolide, as well as fluoroquinolone, resistance. On the other hand, cells became more susceptible to novobiocin and β-lactam cloxacillin. We urge the control of, and adjustments to, treatments with antibiotics and the need for nove ...[more]

PMID: 35311521

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Project description:UnlabelledThe AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.ImportanceThe AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

Dataset Information

Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles.

Publications

Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles.

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