Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, Kx = 6.8 × 105. Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and 1H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of koff = 29 s-1 and kon = 2.4 × 104 M-1 s-1, respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to Kx′ = 2.2 × 104 for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, kp = 1.2 × 10-3 s -1, with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and 1H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry