Research performed at Diamond Light Source has helped to understand how a bacterium responsible for many hospital-acquired infections is able to resist one of the most widely used antiseptic agents. By using ultraviolet light to perform circular dichroism measurements of the proteins within the cells, the scientists were able to characterise a previously unknown method by which the antiseptic could be actively transported out of the cell.
The work focused on the antiseptic chlorhexidine, which is found in dental mouthwash and many antiseptic skin creams, and is widely used in hospitals as an antiseptic and disinfectant. Increasingly bacteria are showing a resistance to chlorhexidine, in particular Acinetobacter baumannii (A. baumannii). The researchers were able to combine a wide array of data to reveal a previously uncharacterised efflux pump (the mechanism by which toxic substances or antibiotics are moved out of a cell).
The work involved contributors from the Department of Chemistry and Biomolecular Sciences, Macquarie University, Australia; the Astbury Centre for Structural Molecular Biology and School of Biomedical Sciences, University of Leeds, United Kingdom; and the School of Biological Sciences, Flinders University, Australia.
Image: Hypothesised operation of the AceI efflux protein
The work first identified a gene in A. baumannii that mediated chlorhexidine resistance. The membrane protein, AceI, encoded by the gene was identified and purified. Then the B23 beamline was used to perform UV circular dichroism on the protein.
The measurements taken at Diamond were able to demonstrate that chlorhexidine bound directly to the AceI protein. The secondary structure and thermal stability of the AceI protein and a mutant were also observed using the beamline, with their structures largely shown to be α-helical.
Professor Peter Henderson of the University of Leeds, one of the lead scientists on the project says, "Efflux is one of many mechanisms by which drug resistance occurs, but it is also the least characterised. The Diamond beamline gave us a clear confirmation of this direct binding, and was a key element in getting our research into the public domain. What is more, these findings could very well be used in the future design of novel anti-bacterials."
To find out more about using the B23 beamline, or to discuss potential applications, please contact Dr Rohanah Hussain: firstname.lastname@example.org
Hassan, K. A., Jackson, S. M., Penesyan, Patching, S. G., Tetu, S. G., Eijkelkamp, B. A., Brown, M. H., Henderson, P. J. F., Paulsen, I. T. Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins. PNAS 110. Issue 50, 20254–20259 (2013), doi:10.1073/pnas.1317052110