title: Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins.
abstract: Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other γ- or β-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [(14)C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia.
authors.forename: Karl A
authors.initials: KA
authors.lastname: Hassan
authors.forename: Scott M
authors.initials: SM
authors.lastname: Jackson
authors.forename: Anahit
authors.initials: A
authors.lastname: Penesyan
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.forename: Sasha G
authors.initials: SG
authors.lastname: Tetu
authors.forename: Bart A
authors.initials: BA
authors.lastname: Eijkelkamp
authors.forename: Melissa H
authors.initials: MH
authors.lastname: Brown
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: Ian T
authors.initials: IT
authors.lastname: Paulsen
mesh_terms: D040981:Acinetobacter baumannii / Q000235:genetics* / Q000378:metabolism; D002710:Chlorhexidine / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005453:Fluorescence; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D046228:Microarray Analysis; D005810:Multigene Family / Q000235:genetics*; D016296:Mutagenesis
chemical_list: D026901:Membrane Transport Proteins; D002710:Chlorhexidine
keywords: drug resistance; membrane transport; opportunistic pathogen