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Proc Natl Acad Sci USA. Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins
[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]
Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins
Karl A. Hassan<SUP>a</SUP>, Scott M. Jackson<SUP>b</SUP>, Anahit Penesyan<SUP>a</SUP>, Simon G. Patching<SUP>b</SUP>, Sasha G. Tetu<SUP>a</SUP>, Bart A. Eijkelkamp<SUP>c</SUP>, Melissa H. Brown<SUP>c</SUP>, Peter J. F. Henderson<SUP>b</SUP>,<SUP>1</SUP>, and Ian. T. Paulsen<SUP>a</SUP>,<SUP>1</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia; <SUP>b</SUP>Astbury Centre for Structural Molecular Biology and School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom; and <SUP>c</SUP>School of Biological Sciences, Flinders University, Bedford Park, SA 5042, Australia
Edited by Helen I. Zgurskaya, University of Oklahoma, Norman, OK, and accepted by the Editorial Board October 31, 2013 (received for review September 11, 2013)
Significance
Drug resistance is an increasing problem in clinical settings with some bacterial pathogens now resistant to virtually all available drugs. Chlorhexidine is a commonly used antiseptic and disinfectant in hospital environments, and there is increasing resistance to chlorhexidine seen in some pathogenic bacteria, such as Acinetobacter baumannii. This paper examines the global gene expression of A. baumannii in response to chlorhexidine exposure and identifies a gene that we demonstrate to mediate chlorhexidine resistance. Biochemical investigation reveals that this gene encodes a previously uncharacterized type of drug efflux pump that actively transports chlorhexidine out of the cell.
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 [<SUP>14</SUP>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.
drug resistance - membrane transport - opportunistic pathogen
Footnotes
<SUP>1</SUP>To whom correspondence may be addressed. E-mail: ian.paulsen@mq.edu.au or p.j.f.henderson@leeds.ac.uk.
Author contributions: K.A.H., M.H.B., P.J.F.H., and I.T.P. designed research; K.A.H., S.M.J., A.P., S.G.P., S.G.T., and B.A.E. performed research; K.A.H., S.M.J., S.G.P., P.J.F.H., and I.T.P. analyzed data; and K.A.H. and I.T.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. H.I.Z. is a guest editor invited by the Editorial Board.
Data deposition: Transcriptomic data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE51525).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1317052110/-/DCSupplemental.
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Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins
Karl A. Hassan<SUP>a</SUP>, Scott M. Jackson<SUP>b</SUP>, Anahit Penesyan<SUP>a</SUP>, Simon G. Patching<SUP>b</SUP>, Sasha G. Tetu<SUP>a</SUP>, Bart A. Eijkelkamp<SUP>c</SUP>, Melissa H. Brown<SUP>c</SUP>, Peter J. F. Henderson<SUP>b</SUP>,<SUP>1</SUP>, and Ian. T. Paulsen<SUP>a</SUP>,<SUP>1</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia; <SUP>b</SUP>Astbury Centre for Structural Molecular Biology and School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom; and <SUP>c</SUP>School of Biological Sciences, Flinders University, Bedford Park, SA 5042, Australia
Edited by Helen I. Zgurskaya, University of Oklahoma, Norman, OK, and accepted by the Editorial Board October 31, 2013 (received for review September 11, 2013)
Significance
Drug resistance is an increasing problem in clinical settings with some bacterial pathogens now resistant to virtually all available drugs. Chlorhexidine is a commonly used antiseptic and disinfectant in hospital environments, and there is increasing resistance to chlorhexidine seen in some pathogenic bacteria, such as Acinetobacter baumannii. This paper examines the global gene expression of A. baumannii in response to chlorhexidine exposure and identifies a gene that we demonstrate to mediate chlorhexidine resistance. Biochemical investigation reveals that this gene encodes a previously uncharacterized type of drug efflux pump that actively transports chlorhexidine out of the cell.
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 [<SUP>14</SUP>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.
drug resistance - membrane transport - opportunistic pathogen
Footnotes
<SUP>1</SUP>To whom correspondence may be addressed. E-mail: ian.paulsen@mq.edu.au or p.j.f.henderson@leeds.ac.uk.
Author contributions: K.A.H., M.H.B., P.J.F.H., and I.T.P. designed research; K.A.H., S.M.J., A.P., S.G.P., S.G.T., and B.A.E. performed research; K.A.H., S.M.J., S.G.P., P.J.F.H., and I.T.P. analyzed data; and K.A.H. and I.T.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. H.I.Z. is a guest editor invited by the Editorial Board.
Data deposition: Transcriptomic data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE51525).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1317052110/-/DCSupplemental.
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