Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.

Funding: This work is supported by National Institutes of Health R21 AI088123 to Y.S. and NIH R01 AI054830 to L.Z. A.C. is a fellow in the Infection and Immunity Training Program (NIH T32 AI55413). This project was supported in part by facilities and resources of the Michael E. DeBakey VA Medical Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 Carlson-Banning et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Understanding how ciclopirox functions is important for uncovering additional repurposed clinical applications and could aid future ciclopirox derivatization. Here we demonstrate the effectiveness of ciclopirox against multidrug-resistant (MDR) Escherichia coli, K. pneumoniae, and A. baumannii clinical isolates. We show that ciclopirox affects the galactose salvage pathway, a novel mechanism of action for this drug.

In spite of the multiple potential uses of ciclopirox, neither its drug target nor its mechanism of action is known. Genetic analyses in Saccharomyces cerevisiae and Candida albicans have been performed in attempts to understand how ciclopirox olamine functions, as this compound does not inhibit ergosterol biosynthesis like other antifungal agents [16] – [18] . In S. cerevisiae, a forward genetic screen identified fourteen mutants that were more susceptible to ciclopirox olamine. Mutations were identified in genes encoding proteins involved in DNA replication, DNA repair, cellular transport, oxidative stress, and signal transduction [16] . Results from S. cerevisiae, however, may not reflect the target of the drug because ciclopirox olamine only weakly inhibits S. cerevisiae growth [16] . A better experimental organism, the human pathogen, C. albicans, is susceptible to low µM ciclopirox olamine concentrations. Microarray analyses of ciclopirox olamine-treated C. albicans revealed gene expression level changes similar to those exhibited in iron-deprived conditions [17] , [18] . Additionally, iron added to growth medium ameliorates ciclopirox olamine inhibition [17] – [19] . Together these data formed the basis for the model that ciclopirox olamine inhibits cells through general iron chelation, but that oxygen accessibility and additional iron-independent mechanisms may also influence ciclopirox olamine efficacy [10] , [18] .

To identify new antimicrobial agents with novel targets, our effort in targeting 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) yielded an N-hydroxypyridinone compound that shows broad-spectrum antibacterial activity [9] . A substructure based literature search led to the identification of ciclopirox, which has more potent antibacterial activity. Ciclopirox is an off-patent, topical antifungal drug developed almost forty years ago that is appealing to repurpose as an antibiotic because of its excellent safety profile. No fungal resistance has been identified in over twenty years of clinical use [10] . Indeed, others have suggested repurposing ciclopirox as an anti-human immunodeficiency virus drug [11] , an agent to protect against mitochondrial damaged cells [12] , and a way to enhance diabetic wound healing [13] . Additionally, ciclopirox is currently in a Phase I clinical trial for treatment of multiple myeloma [14] , [15] .

Antibiotic-resistant gram-negative infections will continue to cause serious health problems because few of the antibiotics presently in development are effective against them [4] , [5] . In addition, most new antibiotics are derivatives of existing drugs and, thus, have bacterial targets already under strong selection to develop resistance. The recent outbreak of carbapenem-resistant Klebsiella pneumoniae at the U.S. National Institutes of Health Clinical Center that caused six patient deaths illustrates how quickly these outbreaks spread and that vigilant precautions are needed for containment [6] . Identification of new antimicrobial agents, particularly those that affect novel targets, is needed to provide effective treatment options. Developing novel antimicrobial agents, however, usually takes a decade or more and costs millions of dollars. Repurposing already approved therapies for alternative uses saves both time and money [7] . Already such a strategy was used to find off-patent drugs to repurpose against antibiotic-resistant Acinetobacter baumannii [8] .

The World Health Organization lists antibiotic-resistant bacterial infections as an important public health problem [1] . In the U.S. alone, two million patients contract hospital-acquired infections, and 50–70% of these infections are antibiotic-resistant [2] , resulting in the deaths of approximately 99,000 patients each year [3] . Longer hospital stays and increased morbidity and mortality as a consequence of antibiotic resistance translate to yearly estimated costs as high as $10 billion [3] . Gram-negative infections are particularly problematic and account for 47% of ventilator-associated pneumonias, 45% of urinary tract infections, and 70% of all intensive care unit infections [4] . If not appropriately treated, these infections can progress to sepsis and death.

Results

Iron Supplementation Prevents Ciclopirox-mediated E. coli Growth Inhibition Iron chelation is a proposed mechanism of action for ciclopirox [17], [19]. Physiochemical studies showed that ciclopirox forms metal complexes with Mg2+, Ca2+, Cu2+, Fe2+, Zn2+, and Mn2+ and that these complexes display a wide range of water solubility and lipophilicity [25]. Researchers working with C. albicans demonstrated that medium supplemented with FeCl 3 prevented ciclopirox olamine-mediated inhibition of fungal growth [17]–[19]. We tested whether addition of iron or other divalent cations could rescue E. coli exposed to ciclopirox. We measured growth of E. coli isolate ATCC® 25922™ in inhibitory ciclopirox concentrations supplemented with increasing concentrations of FeCl 3 , MgCl 2 , ZnCl 2 , or NiCl 2 . Compared to no added metals (Figure 3A), 5 µM FeCl 3 allowed some bacterial growth (Figure 3B), while concentrations of 50 µM FeCl 3 rescued bacterial growth in ciclopirox-treated cultures (Figure 3C). The other metals did not rescue growth, even at concentrations of 500 µM (Figure 3D, 3E, and 3F). These data show that ciclopirox inhibition can be ameliorated with high concentrations of iron. During a bacterial infection, however, the human host defense system actively sequesters free iron to limit pathogen growth [26]. Indeed, unbound ferric iron concentrations in plasma, lymph, and external secretions of milk and bronchial mucus have been reported to be ∼10−18 M, which is far below the 50 µM needed to rescue bacterial growth from ciclopirox inhibition [27]. While ciclopirox iron chelation would help attenuate infections, other iron-independent mechanisms of action may be involved or more important under physiological conditions.

Effect of Ciclopirox on the Ability of E. coli to Survive Hydrogen Peroxide Exposure It was previously found that ciclopirox olamine sensitized C. albicans to H 2 O 2 exposure [17], [18]. In addition, the expression of genes, like catalase, that detoxify reactive oxygen species (ROS) have been linked to ciclopirox olamine inhibition of C. albicans and for bactericidal antibiotic function [17], [18], [28]. We tested whether E. coli exposed to ciclopirox became sensitized to subsequent H 2 O 2 exposure. E. coli isolate ATCC® 25922™ was grown with increasing sub-inhibitory concentrations of ciclopirox to mid-logarithmic phase and then exposed to either water or H 2 O 2 . Chloramphenicol served as a positive control because it sensitizes cells to H 2 O 2 [29]. Whereas preincubation with 1 µg/ml chloramphenicol reduced CFUs following H 2 O 2 exposure by more than 10-fold compared to water (Figure 4; p = 0.008), preincubation with ciclopirox had no effect. This lack of sensitization is consistent with the mechanism of action of some bacteriostatic antibiotics [28] and agrees with the above time-kill curve results at sub-inhibitory ciclopirox concentrations. These data contrast to the C. albicans data, most likely because ciclopirox is fungicidal at sub-inhibitory concentrations. However for bacterial growth, hydrogen peroxide exposure does not appear to synergize with sub-inhibitory ciclopirox concentrations. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Effects of hydrogen peroxide exposure on bacterial response to ciclopirox. ATCC® 25922™ E. coli were grown to mid-logarithmic phase (OD 600 = 0.4) and then subjected to either water or 5 µM H 2 O 2 for 20 minutes. Colony forming units (CFUs) were measured per 1 ml of E. coli grown with either 0, 2.5, 5.0, or 7.5 µg/ml ciclopirox or 1.0 µg/ml chloramphenicol. Each triangle represents a result from an independent culture and the gray bar shows the average. Statistical significance was measured using Student’s t-test. https://doi.org/10.1371/journal.pone.0069646.g004

Overexpression of galE Rescues Ciclopirox Inhibition Microarray analyses revealed that 25 of the 6,039 C. albicans genes were up-regulated and 21 were down-regulated with ciclopirox olamine incubation [18]. The majority of the up-regulated genes were involved in iron metabolism, and the rest included genes that encode Rbt5 glycosylphosphatidylinositol (GPI)-like proteins, transcription factors, an RNA binding protein, NADP-glutamate-dehydrogenase, superoxide dismutase Sod4, and two unknown proteins. The down-regulated genes included those encoding proteins involved with general stress responses, cell elongation, phosphate uptake, catalase, and many of unknown function. These microarray data indicated that some of these genes might encode targets of ciclopirox or proteins that might cause resistance to ciclopirox. Culturing C. albicans for six months with sub-inhibitory ciclopirox olamine concentrations, however, did not yield a resistant mutant [17]. With more genetic tools available for E. coli than C. albicans, we reasoned that an overexpression suppression screen in E. coli would identify pathways affected by ciclopirox. To identify genes that, when overexpressed, rescued E. coli growth at otherwise restrictive ciclopirox concentrations, we transformed pools of plasmids from the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) pCA24N ORF library into TransforMax™ EC100™ Electrocompetent E. coli. Transformed cells were grown under selective ciclopirox concentrations of 7.5 µg/ml. 540 candidate transformants were streaked across agar containing a gradient of 0 to 18 µg/ml ciclopirox. MICs were then measured for 50 candidates that grew at higher ciclopirox concentrations than the other candidates. Only six of these transformants had ciclopirox MICs greater than the parent strain. The pCA24N plasmids from these six transformants were purified and the ORF was sequenced using previously described primers [30]. The purified plasmids were used to transform the parent strain, and the increase in ciclopirox MIC was confirmed. For all six candidates, the sequenced ORF was galE, which encodes UDP-glucose 4-epimerase. We confirmed that overexpression of galE rescued growth at previously restrictive ciclopirox concentrations (Figure 5A, 5B, and 5C). These data indentify GalE or the GalE pathway as a potential target of ciclopirox. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Effect of GalE on ciclopirox inhibition of bacterial growth. Growth curves for three independent cultures of TransforMax™ EC100™ Electrocompetent E. coli (A) without overexpression plasmid, (B) with empty plasmid pCA24N, or (C) with pCA24N-galE grown with the indicated ciclopirox concentrations. Error bars are the standard deviation from the mean. (D) Schematic representation of GalE epimerization of UDP-galactose to UDP-glucose coupled to the activity of UDP-glucose dehydrogenase (UGD). (E) Using the assay schematized in D, the average rate of NADH formation with or without ciclopirox was measured three independent times per ciclopirox concentration. Error bars are the standard deviation from the mean. https://doi.org/10.1371/journal.pone.0069646.g005

Ciclopirox does not Inhibit Purified GalE In E. coli and other bacteria, GalE epimerizes UDP-galactose and UDP-glucose. To test whether ciclopirox directly inhibits GalE, we purified 6xHis-GalE from E. coli DH5α cells transformed with the pCA24N-galE overexpression plasmid. Using previously described assays depicted in Figure 5D [31], [32], we coupled GalE activity to purified UDP-glucose dehydrogenase (UGH). UDP-galactose was used as a substrate for GalE. After GalE epimerizes UDP-galactose into UDP-glucose, UGH then converts UDP-glucose into UDP-glucuronic acid with the concomitant release of two molecules of NADH. The production of NADH is spectrophotometrically measured at 340 nm. To assess whether ciclopirox could inhibit the epimerization of UDP-galactose, we measured NADH production in the presence of increasing ciclopirox concentrations (Figure 5E). For these experiments, ciclopirox was dissolved in 0.1 mM NaOH because we found that dimethyl sulfoxide (DMSO) inhibits GalE activity (Figure S1). For all reactions, the concentration of UDP-galactose used was 50 µM, which is below the published K m (100–200 µM) for E. coli GalE. As a control, we ensured that NADH production was observed only when UDP-galactose was added; there was no spontaneous NADH formation. As an additional control, we verified that ciclopirox did not inhibit the coupled enzyme, UGD (Figure S2). GalE epimerization of UDP-galactose was not affected by ciclopirox, even at concentrations of 500 µM (Figure 5E). Although this assay does not address the possibility that ciclopirox may affect GalE epimerization of UDP-glucose into UDP-galactose, this reaction is less favored [33]. These data suggest that other targets in the GalE pathway are affected by ciclopirox, or that the nucleotide-sugars GalE produces help the bacteria cope with ciclopirox-induced cellular stresses.

Effect of Mutations in the Galactose Salvage and Lipopolysaccharide Biosynthesis Pathways on Ciclopirox MICs Regulation of nucleotide-sugar concentrations is required for organisms to adjust to environmental stresses [34]–[36]. Depending on cellular needs, the galactose salvage pathway either metabolizes galactose for energy or uses galactose to build metabolic intermediates for lipopolysaccharide and exopolysaccharide construction (Figure 6A and 6B) [36]–[38]. When galactose or lactose is unavailable, GalE is required to synthesize UDP-galactose; however, when galactose is the sole carbon source, GalE synthesizes UDP-glucose, which is then converted to glucose-1-phosphate by GalU to be used in glycolysis [35], [36]. Thus, GalE is essential when E. coli is grown in galactose medium. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Effect of ciclopirox on strains involved in the galactose and lipopolysaccharide biosynthesis pathways. (A) Schematic representations of the galactose salvage pathway, (B) LPS biosynthesis pathway proteins for E. coli K-12 strain. Note the O-antigen is not present in K-12 strains, but is depicted for reference for where the O-antigen can attach. Abbreviations include fructose (Fru), glucose (Glc), galactose (Gal), heptose (Hep), N-acetylglucosamine (GlcN), 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo), and phosphate (P). (C) Ciclopirox MICs for Keio parent, BW25113, and single gene Keio deletion strains were measured in three independent cultures. These genes encode enzymes involved in the galactose metabolism and LPS biosynthesis pathways. Student’s t-test was used to assess significance. https://doi.org/10.1371/journal.pone.0069646.g006 GalE is unique among the other members of the galactose salvage and LPS pathways. Even when grown in medium with other sugars, overexpression of most of the other galactose salvage pathway genes (galK, galM, galP, galU, galR, galS, and galT) are lethal for bacteria. Only overexpression of glf or galE is not lethal (Figure 6A) [30], [39]. The non-essential LPS biosynthesis genes, rfaB, rfaC, rfaF, rfaG, rfaI, rfaL, rfaP, rfaQ, rfaY, and rfaZ are also toxic when overexpressed in any sugar source, while the rfaD, rfaJ, or rfaS genes are not (Figure 6B) [30], [39]. However, in glucose medium, deletions of these same galactose metabolism or LPS biosynthesis genes are not lethal to bacteria. Bacteria harboring deletions for proteins that promote cell survival upon ciclopirox exposure should make these bacteria more susceptible to the drug. Thus, ciclopirox MICs were measured in strains with deletion of genes known to be involved in the galactose salvage pathway (ΔgalE, ΔgalK, ΔgalM, ΔgalU, ΔgalT, Δglf and Δugd), the regulators of the galactose salvage pathway (ΔgalR and ΔgalS), the glycolysis pathway (Δpgi and Δpgm), or the lipopolysaccharide biosynthesis pathway (ΔrfaB, ΔrfaC, ΔrfaD, ΔrfaF, ΔrfaG, ΔrfaI, ΔrfaJ, ΔrfaL, ΔrfaP, ΔrfaQ, ΔrfaS, and ΔrfaZ). Compared to the Keio parent strain, BW25113, 17/26 deletion mutants had altered average ciclopirox MICs; eight of these (ΔgalE, ΔgalU, ΔgalR, Δglf, ΔrfaB, ΔrfaC, ΔrfaI, and ΔrfaQ) were statistically significantly more susceptible to ciclopirox (Figure 6C; p = 0.05). The ΔrfaC, ΔrfaD, ΔrfaG, ΔrfaP, ΔrfaQ, and ΔrfaY LPS pathway mutants had previously been shown to display enhanced sensitivity to other antibiotics compared to the parent BW25113 strain, but the galactose salvage pathway deletions were not affected by the tested antibiotics [40]. Therefore, whereas it was not surprising that the ΔrfaC and ΔrfaQ deletion mutant strains were more susceptible to ciclopirox, the other mutants that had increased sensitivity to ciclopirox were surprising. To determine whether the decrease in ciclopirox MIC in ΔgalE, ΔgalU, ΔrfaB, and ΔrfaI mutants was specific to ciclopirox or could be observed for other antibiotics, we measured ampicillin, aztreonam, chloramphenicol, and ciprofloxacin MICs (Table S3). Antibiotic MICs for ΔrfaJ mutants were also measured. Compared to BW25113, there were no significant susceptibility changes to ampicillin or ciprofloxacin. The ΔrfaI and ΔrfaJ strains were less susceptible to aztreonam, and the ΔgalE and ΔrfaB strains were slightly more susceptible to chloramphenicol, but ΔrfaJ strains were less susceptible to chloramphenicol. These data suggest that the ΔgalE, ΔgalU, ΔrfaB, and ΔrfaI mutants are specifically more susceptible to ciclopirox, but not to other antibiotics tested. Aside from RfaC and RfaQ, which are involved in synthesis of the inner LPS core, GalE, GalU, RfaB, and RfaI are all involved in synthesis of the outer LPS core, and all of these proteins either synthesize or utilize UDP-glucose or UDP-galactose. RfaJ is also involved in synthesis of the outer LPS and uses UDP-glucose, but ΔrfaJ mutants did not have altered ciclopirox susceptibility compared to the parental strain. These data suggest that LPS biosynthesis processes affected by ciclopirox could be dependent on the specific sugars or sugar linkages present. Of the remaining deletion strains tested, five (ΔgalF, Δpgm, ΔrfaF, ΔrfaP, and ΔrfaG) were less susceptible to ciclopirox than the parental strain. The deletions mutants that were the least susceptible to ciclopirox, ΔrfaF, ΔrfaG, and ΔrfaP, encode proteins that synthesize the inner LPS core [41]. RfaF adds the first glucose group and RfaG adds heptose II to the developing inner LPS core, and deletions of these genes results in no outer core [41]. If no outer core is formed, then there is less demand for UDP-glucose or UDP-galactose. Instead of adding sugars to the inner core, RfaP phosphorylates heptose I and can affect RfaY and RfaQ functions [42]. While ΔrfaP mutants form both inner and outer LPS cores, the lack of phosphorylation affects overall membrane charge and surface hydrophobicity [41]. E. coli utilizes such membrane modification to resist antibiotics, such as polymyxin, and this resistance is clinically relevant [43], [44]. It is possible that changes in the membrane surface charge alters ciclopirox membrane permeability.

Effect of Ciclopirox on LPS Formation GalE is involved with LPS [45]–[47] and exopolysaccharide (EPS) formation [38], [48]. Studies in C. albicans have shown that ciclopirox alters the structure of cell membranes [17], [49], [50]. Indeed, 97% of ciclopirox administered to C. albicans was bound to cell membranes and organelles, with very little drug in the cytoplasm [50]. That we found ciclopirox MICs altered in strains deleted for genes responsible for LPS biosynthesis raises the possibility that ciclopirox alters LPS formation. To test this possibility, overnight cultures of the E. coli ATCC® 25922™ isolate or MG1655 strain were spread onto agar without or with a sub-inhibitory concentration of ciclopirox. After 24 hours, LPS was purified and subjected to SDS-PAGE as described [51] (Figure 7). Of the LPS bands characteristic of the ATCC® 25922™ isolate [52], ciclopirox reduced the concentrations of the highest molecular weight band (∼20 kDa) as well as the O-antigen bands (37–50 kDa), as indicated by the arrows (Figure 6). LPS isolated from MG1655, which lacks an O-antigen, was unchanged in the presence of ciclopirox. The LPS changes mediated by ciclopirox in the ATCC® 25922™ isolate were seen six independent times. These data may indicate a subtle mode of ciclopirox action: LPS is formed but with altered composition, perhaps as a consequence of which nucleotide-charged sugars are available. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. Effect of ciclopirox on E. coli LPS structure. LPS was purified from E. coli clinical isolate ATCC®25922™ or K-12 strain MG1655 that had been either incubated with 9 µg/ml ciclopirox as indicated. LPS was subjected to 12.5% Tris-glycine-SDS-PAGE. Lanes 1 and 2 are LPS from two independent LPS purifications. This result was repeated six times with the same results. https://doi.org/10.1371/journal.pone.0069646.g007