Published on Web 01/25/2005
Membrane Dynamics of the Amphiphilic Siderophore, Acinetoferrin Minkui Luo, Evgeny A. Fadeev, and John T. Groves* Contribution from the Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08540 Received September 22, 2004; E-mail:
[email protected] Abstract: 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.
Introduction
Although iron is one of the most abundant elements on earth, its microbial bioavailability is limited due to the extremely low solubility of iron(III) oxides under aqueous aerobic conditions.1,2 To surmount this problem, most bacteria secrete siderophores, small organic molecules with a high affinity for iron(III).3,4 Among the hundreds of known siderophores, there is a small subfamily of amphiphilic siderophores that typically contain a hydrophilic iron-binding headgroup and one or two hydrophobic side chains. The first amphiphilic siderophores to be identified were the mycobactins.5 Several classes of amphiphilic siderophores have been isolated from a large number of terrestrial bacteria.6-11 It was recently reported that marine microorganisms also adopt similar siderophore-mediated strategy to overcome (1) Albrecht-Gary, A. M.; Crumbliss, A. L. Met. Ions Biol. Syst. 1998, 35, 239-327. (2) Boukhalfa, H.; Crumbliss, A. L. Biometals 2002, 15, 325-339. (3) (a) Ratledge, C.; Dover, L. G. Annu. ReV. Microbiol. 2000, 54, 881-941. (b) Raymond, K. N.; Dertz, E. A.; Kim, S. S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3584-3588. (c) Bluhm, M. E.; Kim, S. S.; Dertz, E. A.; Raymond, K. N. J. Am. Chem. Soc. 2002, 124, 2436-2437. (4) (a) Roosenberg, J. M.; Lin, Y. M.; Lu, Y.; Miller, M. J. Curr. Med. Chem. 2000, 7, 159-197. (b) Bergeron, R. J.; McManis, J. S.; Franklin, A. M.; Yao, H.; Weimar, W. R. J. Med. Chem. 2003, 46, 5478-5483. (c) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Bussenius, J.; Smith, R. E.; Weimar, W. R. J. Med. Chem. 2003, 46, 1470-1477. (d) Bergeron, R. J.; McManis, J. S.; Bussenius, J.; Brittenham, G. N.; Wiegand, J. J. Med. Chem. 1999, 42, 2881-2886. (5) Francis, J.; Macturk, H. M.; Madinaveitia, J.; Snow, G. A. Biochem. J. 1953, 55, 596-607. 1726
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iron deficiency in the oceans.12 Three suites of amphiphilic siderophores, marinobactins, aquachelins, and amphibactins, with lipopeptide-like structures were characterized from different oceanic strains.12,14 Thus, it is apparent that amphiphilic siderophores play important biological roles in bacterial ironacquisition in a variety of iron-deficient circumstances. Acinetoferrin (Af as indicated in Figure 1) is a citrate-based amphiphilic siderophore produced by Acinetobacter haemolyticus,7 the most prevalent proteolytic species of the ubiquitous (6) Lynch, D.; O’Brien, J.; Welch, T.; Clarke, P.; Cuiv, P. O.; Crosa, J. H.; O’Connell, M. J. Bacteriol. 2001, 183, 2576-2585. (7) Okujo, N.; Sakakibara, Y.; Yoshida, T.; Yamamoto, S. Biometals 1994, 7, 170-176. (8) Persmark, M.; Pittman, P.; Buyer, J. S.; Schwyn, B.; Gill, P. R.; Neilands, J. B. J. Am. Chem. Soc. 1993, 115, 3950-3956. (9) Risse, D.; Beiderbeck, H.; Taraz, K.; Budzikiewicz, H.; Gustine, D. Z. Naturforsch., C: Biosci. 1998, 53, 295-304. (10) (a) Stephan, H.; Freund, S.; Beck, W.; Jung, G.; Meyer, J. M.; Winkelmann, G. Biometals 1993, 6, 93-100. (b) Stephan, H.; Freund, S.; Meyer, J. M.; Winkelmann, G.; Jung, G. Liebigs Ann. Chem. 1993, 43-48. (11) Groves, J. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3569-3574. (12) (a) Martinez, J. S.; Carter-Franklin, J. N.; Mann, E. L.; Martin, J. D.; Haygood, M. G.; Butler, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 37543759. (b) Martinez, J. S.; Zhang, G. P.; Holt, P. D.; Jung, H. T.; Carrano, C. J.; Haygood, M. G.; Butler, A. Science 2000, 287, 1245-1247. (13) Levitt, M. H. Spin Dynamics: Basics of the Nuclear Magnetic Resonance; John Wiley & Sons: Chichester, New York, Weinheim, Brisbane, Singapore, Toronto, 2001. (14) (a) Xu, G. F.; Martinez, J. S.; Groves, J. T.; Butler, A. J. Am. Chem. Soc. 2002, 124, 13 408-13 415. (b) Xu, G. F. Synthesis and Analyses of Materials at Interfaces: From Organic Soft Materials to Inorganic Hard Materials. Ph.D. Thesis, the Department of Chemistry, Princeton University, 2002, Chapter 8. 10.1021/ja044230f CCC: $30.25 © 2005 American Chemical Society
Membrane Dynamics of Acinetoferrin
Figure 1. Structures of citrate-based siderophores.
Acinetobacter genus.16 An increasing number of Acinetobactermediated hospital infections and outbreaks have been reported recently since these organisms are highly resistant to many antibiotics in common use, including β-lactams, macrolides, and quinolones.7,17,18 Af secreted by Acinetobacter and its derivative, acinetoferrin imide, is able to cross-feed iron to Mycobacterium paratuberculosis and Mycobacterium tuberculosis, two pathogenic strains that are responsible for Crohn’s disease and lethal tuberculosis in humans, respectively.19 The emergence of these infections and their corresponding multidrug resistances make it important to understand the Af-mediated iron transport. (15) In a previous paper,14 the equation kobs/kw ) (R - 1)[(Kx × [vesicle])/(Kx × [vesicle] + [water])] + 1 was used instead of eq 2. The additional term k′ × [vesicle] in eq 2 is to correct for the variation in the ionic strength with varied lipid concentrations. As shown in Figure S3, the relative ironacquisition rates (kobs/kw) dropped rapidly after initial addition of lipid vesicles (
