An efficient and economical assay to screen for triclosan-resistant Staphylococcus aureus
Triclosan is an effective inhibitor for an essential bacterial enzyme, the enoyl acyl carrier protein reductase (ENR/FabI), in fatty acid biosynthesis. Due to its value in inhibiting bacterial growth, including methicillin resistant Staphylococcus aureus (S. aureus) (MRSA) strains, triclosan has been widely used to prevent infections. Species resistance to triclosan can arise from mutations in ENR/FabI. For example, triclosan-resistant S. aureus mutants have been reported, suggesting that other S. aureus mutants may also survive triclosan treatment, and need to be identified.
The standard method for identifying a triclosan-resistant mutant is to perform an enzyme activity inhibition assay. However, the enzyme assays for S. aureus are challenging due to the need to prepare the substrate, enoyl acyl carrier protein. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use protein-linked substrates, to determine the binding ability of triclosan to ENR/FabI active site, and thus can be used for screening for triclosan-resistant mutants. This method may also be applied to select effective triclosan analogues that inhibit ENR/FabI activity.
By following the fluorescence intensity of a probe (Sypro Orange) in S. aureus ENR/FabI sample as a function of temperature, a characteristic transition temperature, Tm, is obtained from the protein unfolding profile. When a compound binds to a protein molecule, it will stabilize the protein to give a higher transition temperature. We determine the transition temperatures of S. aureus ENR/FabI in the absence and presence of triclosan and NADP+. If triclosan and NADP+ do not bind ENR/FabI, the Tm is about 40 oC. If triclosan and NADP+ bind ENR/FabI, the Tm is about 60 oC. A 20 oC temperature shift is detected. The thermal shifts are observed only when both triclosan and NADP+ are present, but not when only one is present. Thus the binding we observed is the active-site binding.
Robel D. Demissie, Pauline Kabre, Micheal L. Tuntland, Leslie W.-M. Fung
Department of Chemistry, University of Illinois at Chicago
An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.
Demissie RD, Kabre P, Tuntland ML, Fung LW
J Biomol Screen. 2015 Nov 4