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The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent

Ehud Banin*†, Alina Lozinski‡, Keith M. Brady§, Eduard Berenshtein¶, Phillip W. Butterfield , Maya Moshe†, Mordechai Chevion¶, Everett Peter Greenberg*,**††, and Eyal Banin‡**

*Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195; †Institute for Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel; ‡Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel; §Department of Microbiology, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, IA 52242; ¶Department of Cellular Biochemistry and Human Genetics, The Hebrew University of Jerusalem, Jerusalem 91120, Israel;

and Department of Civil and Environmental Engineering, Washington State University, Spokane, WA 99210 Contributed by Everett Peter Greenberg, September 2, 2008 (sent for review June 5, 2008)

The opportunistic pathogen Pseudomonas aeruginosa causes infec- tions that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine- gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic genta- micin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

antimicrobials biofilms chronic infections iron uptake keratitis

Bacteria can exist in a free-living planktonic state and also in a biofilm state. Biofilms are groups of bacteria associated with a surface and embedded in a self-produced extracellular matrix (1, 2). As a consequence of the biofilm lifestyle, bacteria can tolerate exposure to antibiotics; biofilm infections are no- toriously difficult to treat and often impossible to cure (1, 3). Various approaches to inhibit bacterial growth and biofilm formation include novel antibiotics, anti-quorum sensing mole- cules, and modulation of iron availability (4, 5). Iron is an especially intriguing target as it is an essential element for growth of most organisms, including bacteria. In fact, maintaining free iron concentrations at extremely low levels ( 10 18 M) is a first line of host defense against invading pathogens (6, 7).

For Pseudomonas aeruginosa, an opportunistic pathogen that can cause both acute and chronic infections (1, 8), iron is not only a necessary element for growth but also a cue in biofilm formation (9, 10). Thus, interfering with bacterial iron ho- meostasis may serve as a potential therapeutic target that can block P. aeruginosa virulence in both the free-living and biofilm states. Targeting bacterial iron metabolism to treat infections has been explored previously. Published reports describe the use of siderophore-antibiotic conjugates (11, 12) and attempts to re- place the active Fe(III) moiety with a metabolically-inactive metal ion such as Sc(III), In(III), or Ga(III) (4, 13, 14). Both approaches yielded promising results when applied to free-living bacteria. Most recently, Kaneko et al. (4) examined the use of Ga(NO3)3 to eradicate P. aeruginosa biofilm cells. Gallium inhibited P. aeruginosa growth, prevented biofilm formation, and showed bactericidal activity against free-living as well as biofilm cells. Furthermore, Ga was effective in two different experimen- tal animal infections simulating acute lethal pneumonia and a chronic airway biofilm infection.

Here we report a novel approach for delivery of gallium to P. aeruginosa. We use a metallo-complex that combines a strong siderophore, desferrioxamine (DFO) with gallium, a redox- inactive metal ion. Because Ga(III) ligand chemistry shares

similarity with that of Fe(III), it interferes with iron metabolism (4, 14). The DFO-Ga complex was originally designed as an antioxidant that can act by ‘‘push and pull’’ mechanisms, se- questering ferric ions (the siderophore effect) and, in turn, releasing gallium ions that further compete with ferric ions at iron binding sites of proteins (15, 16). Here we exploit the chemical and structural similarities between DFO-Fe and DFO-Ga to interfere with iron homeostasis in P. aeruginosa. We chose DFO as a siderophore carrier of Ga because P. aeruginosa is thought to possess two uptake systems for DFO-Fe (10). Thus DFO-Ga could serve as a Trojan horse that delivers toxic gallium to P. aeruginosa cells via either of the two DFO uptake systems.

Results

DFO-Ga Kills planktonic P. aeruginosa Cells. We first examined the effect of DFO-Ga on planktonic P. aeruginosa in a low iron medium. The minimal inhibitory concentration (MIC) of DFO-Ga under these conditions was 0.032 mM. A similar result was obtained for Ga(III) alone (GaCl3). As expected, DFO did not inhibit growth, even at a concentration of 1 mM. Like many other bacterial species, when in the stationary phase, P. aeruginosa is not effectively eradicated by antibiotics (17), and part of the explanation for biofilm resistance to antibiotic treatment is that a large fraction of the bacterial cells in a biofilm are likely to be in a stationary phase-like state. We thus tested the ability of DFO-Ga to kill stationary phase cells (Fig. 1). After a long (24 h) incubation period, gentamicin (Gm) (10 g per ml, more than 10 times the MIC) reduced P. aeruginosa viability by three log units, while DFO-Ga (1 mM) was 10–100 fold more effective. Because DFO-Ga and Gm exert their antimicrobial effects via different mechanisms, we examined whether they have an additive effect when administered together. The combined treatment resulted in a remarkable six-log reduction in the number of viable cells, suggesting synergy between DFO-Ga and Gm (Fig. 1A).

To examine the role of the previously identified putative DFO receptors (10) in killing by DFO-Ga, we obtained two single mutant strains and generated a double mutant. The mutations were in either PA0470, PA2466, or both genes. We then com- pared the sensitivities of these mutants to Ga and DFO-Ga (Fig. 1B). The single mutants and wild-type were equally sensitive to Ga and DFO-Ga (data not shown), whereas the double mutant

Author contributions: Ehud Banin, A.L., E. Berenshtein, P.W.B., M.M., M.C., E.P.G., and Eyal Banin designed research; Ehud Banin, A.L., K.M.B., E. Berenshtein, P.W.B., and M.M. performed research; Ehud Banin, E. Berenshtein, and M.C. contributed new reagents/ analytic tools; Ehud Banin, A.L., K.M.B., E. Berenshtein, P.W.B., M.M., M.C., E.P.G., and Eyal Banin analyzed data; and Ehud Banin, A.L., M.C., E.P.G., and Eyal Banin wrote the paper.

The authors declare no conflict of interest.

**E.P.G. and Eyal Banin contributed equally to this work.

††To whom correspondence should be addressed. E-mail: epgreen@u.washington.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0808608105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

MICROBIOLOGY

www.pnas.org cgi doi 10.1073 pnas.0808608105

PNAS October 28, 2008 vol. 105 no. 43 16761–16766

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