Spermidine Binding to the Acetinobacter baumannii Efflux Protein AceI Observed by Near-UV Synchrotron Radiation Circular Dichroism Spectroscopy

Abstract

The aim of this work was to test polyamines as potential natural substrates of the Acinetobacter baumannii chlorhexidine efflux protein AceI using near-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. The Gram-negative bacterium A. Baumannii is a leading cause of hospital-acquired infections and an important foodborne pathogen. A. Baumannii strains are becoming increasingly resistant to antimicrobial agents, including the synthetic antiseptic chlorhexidine. AceI (144-residues) was the founding member of the recently recognised PACE family of bacterial multidrug efflux proteins. Using the plasmid construct pTTQ18-aceI(His6) containing the A. baumannii aceI gene directly upstream from a His6-tag coding sequence, expression of AceI(His6) was amplified in E. coli BL21(DE3) cells. Near-UV (250–340 nm) SRCD measurements were performed on detergent-solubilised and purified AceI(His6) at 20 °C. Sample and SRCD experimental conditions were identified that detected binding of the triamine spermidine to AceI(His6). In a titration with spermidine (0–10 mM), this binding was saturable and fitting of the curve for the change in signal intensity produced an apparent binding affinity (KD) of 3.97 ± 0.45 mM. These SRCD results were the first experimental evidence obtained for polyamines as natural substrates of PACE proteins.