Regulation of the expression of the CmeABC efflux pump in Campylobacter jejuni: identification of a point mutation abolishing the binding of the CmeR repressor in an in vitro-selected multidrug-resistant mutant.
A multidrug-resistant mutant of Campylobacter jejuni was selected in vitro using increasing concentrations of enrofloxacin. This mutant accumulated less ethidium bromide than the parental strain, suggesting the participation of active efflux as a resistance mechanism. Inactivation of the cmeB gene confirmed active efflux and indicated the involvement of the CmeABC efflux pump in the multidrug resistance of the mutant. Sequencing of the cmeR-cmeA intergenic region revealed a point mutation in the binding site of the CmeR repressor. Transcriptional lacZ fusions showed an increase of transcription of the cmeABC operon in the multidrug-resistant mutant. Gel mobility shift assays and Surface Plasmon Resonance experiments further indicated a decrease in the affinity of the CmeR for the promoting region of the cmeABC operon consecutive to this mutation. Thus, these results showed that the point mutation was responsible, via a lack of binding of the CmeR repressor, for increased expression of the CmeABC efflux pump and consecutive multidrug resistance.
Cagliero
Cedric
C
Institut National de la Recherche Agronomique, UR1282 Infectiologie Animale et Santé Publique, Nouzilly, France.
Maurel
Marie-Christine
MC
Cloeckaert
Axel
A
Payot
Sophie
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D001693:Biological Transport, Active; D016123:Campylobacter jejuni / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D021901:DNA, Intergenic / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D000077422:Enrofloxacin; D004996:Ethidium / Q000378:metabolism; D024841:Fluoroquinolones / Q000494:pharmacology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009876:Operon; D017354:Point Mutation; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D020349:Surface Plasmon Resonance; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D024841:Fluoroquinolones; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; D000077422:Enrofloxacin; D001616:beta-Galactosidase; D004996:Ethidium