Download Oxygen accessibility and iron levels are critical

JAC
Journal of Antimicrobial Chemotherapy (2005) 55, 663–673
doi:10.1093/jac/dki089
Advance Access publication 14 March 2005
Oxygen accessibility and iron levels are critical factors for
the antifungal action of ciclopirox against Candida albicans
Hans-Christian Sigle1, Sascha Thewes2, Markus Niewerth3, Hans Christian Korting3,
Monika Schäfer-Korting1 and Bernhard Hube2*
1
Institut für Pharmazie, Abteilung für Pharmakologie und Toxikologie, Freie Universität Berlin, Königin-Luise-Str.
2 + 4, D-14195 Berlin, Germany; 2Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany;
3
Dermatologische Klinik und Poliklinik, Universität München, Frauenlobstrabe 9 – 11, D-80337 Munich, Germany
Received 9 December 2004; returned 26 January 2005; revised 2 February 2005; accepted 7 February 2005
Objectives: Ciclopirox is a topical antifungal agent of the hydroxypyridone class whose mode of action
is poorly understood. In order to elucidate the mechanism of action of ciclopirox, we analysed the
growth, cellular integrity, biochemical properties, viability and transcriptional profile of the polymorphic yeast Candida albicans following exposure to this antifungal agent.
Methods: Multiple biochemical assays served to identify factors that were critical for antifungal activity
and to identify proteins whose activities changed in drug-exposed cells. Genome-wide transcriptional
profiling was used to identify genes that were up-regulated in response to the cellular effects of the
drug.
Results: Ciclopirox inhibited growth of C. albicans yeast and hyphal cells in a dose-dependent manner.
This effect was reduced (i) by the addition of iron ions or the metabolic inhibitor 2-deoxy-D -glucose to
growth media, (ii) in media that lacked glucose, and (iii) for cells that were pre-incubated with hydrogen
peroxide or menadione [which caused induction of proteins involved in detoxification of reactive oxygen species (ROS)]. In contrast, cells pre-cultured under poor oxygen conditions (which had decreased
activity of proteins involved in ROS detoxification) were more susceptible to ciclopirox. Treatment with
ciclopirox did not directly cause cell membrane damage and did not change intracellular levels of ATP.
Finally, the transcriptional profiling pattern of drug-treated cells strongly resembled iron-limited
conditions.
Conclusions: These data indicate that metabolic activity, oxygen accessibility and iron levels are critical parameters in the mode of action of ciclopirox olamine.
Keywords: hydroxypyridones, iron metabolism, reactive oxygen species, transcriptional profiling, microarray
Introduction
Hydroxypyridones are effective topically used antifungal agents
with a very broad spectrum against dermatophytes, yeasts, filamentous fungi and bacteria. Despite the fact that hydroxypyridones have been in clinical use for more than 20 years, their
mode of action is poorly understood. This is in contrast to other
antimycotics such as polyenes, azoles, flucytosine or echinocandins.1,2
The minimal inhibitory concentration (MIC) of the hydroxypyridone ciclopirox olamine [ethanolamine salt of 6-cyclohexyl-1-
hydroxy-4-methyl-2(1H)-pyridone, in the following text referred
to as ‘ciclopirox’] was found to be between 0.49 and 3.9 mg/L
for many human pathogenic fungi.3 Iwata & Yamaguchi4 showed
that even low concentrations of ciclopirox inhibit the cellular
uptake of amino acids, potassium and phosphate ions. It was
concluded that the reduced biosynthesis and respiration of ciclopirox-treated cells resulted from the reduced ability to store
these essential components. In contrast to polyenes and azoles,
however, cell membranes were damaged only when very high
concentrations (50 –200 mg/L) of hydroxypyridones were added
to the cells.5 – 7 Furthermore, studies using Candida albicans
..........................................................................................................................................................................................................................................................................................................................................................................................................................
*Corresponding author. Tel: +49-1888-754-2917; Fax: +49-1888-754-2328; E-mail: [email protected]
..........................................................................................................................................................................................................................................................................................................................................................................................................................
663
q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
Sigle et al.
protoplasts and membrane models with ergosterol-containing
liposomes showed that ciclopirox does not directly damage or
interact with cell membranes.4 Since azole-resistant strains are
still susceptible to ciclopirox, the inhibition of ergosterol biosynthesis can be excluded as the principal mode of action of ciclopirox.8 Rilopirox, another hydroxypyridone, reduces the
respiratory activity of Saccharomyces cerevisiae possibly by
influencing the uptake or metabolism of glucose. Hydroxypyridones have also been shown to inhibit the activity of the metal
ion containing enzymes NADH-ubiquinone-oxidoreductase
(complex I) and catalase.9 Since numerous cellular proteins
depend on metal ions as co-factors, binding of metal ions may
be of major importance in the mode of action of hydroxypyridone antimycotics.
In this study, we analysed the effect of ciclopirox on the
growth, cellular integrity and viability of C. albicans cells and
used a series of biochemical assays to identify parameters that
are critical for antifungal activity and to identify proteins whose
activities are modulated in drug-exposed cells. Furthermore, we
used genome-wide transcriptional profiling to identify genes that
may be up-regulated in response to the drug.
Materials and methods
Strains and growth conditions
solution, 90 mL of medium and 10 mL of semi-synchronized cells
(2 108 cells/mL) were added to the wells of a 96-well plate. Final
drug concentrations were between 0.125 and 64 mg/L. Plates were
incubated for 4 h at 378C. Fixation solution (40 mL, 10% SDS/10%
formalin) was added to the wells and the percentage of germ tubes
was counted microscopically (three times 100 randomly chosen
cells). The G-IC30 value was the lowest drug concentration that
caused a 70% reduction in the germ tube formation of the untreated
control.
To analyse the influence of metal ions on the inhibitory effect of
ciclopirox (acid, Aventis) to germ tube formation, we mixed
11.1 mM proline, 22.2 mM glucose and 222 mM of metal ions
(MgSO4, CaCl2, MnCl2, FeCl2 and FeCl3) in PBS pH 7.4. The
ciclopirox stock solution was diluted with PBS pH 7.4 to give concentrations from 0.5 to 64 mg/L. One hundred microlitres of drug
solution, 90 mL of test medium, and 10 mL of semi-synchronized
cells (2 108/mL) were mixed and incubated as described above
and the G-IC30 value was calculated.
To measure the viability of cells exposed to drugs, treated or
untreated cells were diluted and plated on modified Sabouraud
glucose agar or mixed into sterile 408C cooled agar and colony
forming units (cfu) were counted after 48 – 72 h incubation at room
temperature. The cfu was calculated relative to the viability of
untreated cells at the start of the experiments.
Treatment with inhibitors and inducers
Growth and biochemical assays were carried out with the type strain
C. albicans CBS 562 (kindly provided by H. J. Tietz, Berlin,
Germany) and the clinical isolate 5342 ZN (Dermatologische Klinik,
Munich, Germany). Strain CBS 562, a relatively poor germ tube
producer, was preferred to strain 5342 ZN in experiments when
yeast growth was favoured, for example to monitor viability. For
transcriptional profiling experiments, we used the strain SC5314,10
which was used for the Stanford sequencing project (http://wwwsequence.stanford.edu/group/candida). Susceptibility of SC5314 to
ciclopirox was investigated as described recently.11
Semi-synchronized cells were used to standardize growth experiments. To semi-synchronize the cells,12 25 mL of modified Sabouraud
(0.2%)13 glucose medium was inoculated with 2 105 cells/mL and
incubated at 150 rpm for 16 h at room temperature. Cells were
harvested and inoculated (4 106 cells/mL) into fresh medium and
shaken for 24 h at 378C. Cells were again harvested, washed three
times with phosphate-buffered saline (PBS) pH 7.4 and used in
further experiments.
To analyse the susceptibility of C. albicans to antifungal drugs
(IC30), cells were grown in Yeast Nitrogen Base (YNB) (Difco
Laboratories) and Lee’s medium14 and tested in a microdilution test.
Drug solutions (100 mL), 90 mL of medium and 10 mL of semisynchronized cells (107 cells/mL) were added to the wells of a 96well plate. Final drug concentrations were between 0.0037 and
128 mg/L. Controls included medium without any antifungal agent
and medium containing drug solution without fungi. Sealed plates
were incubated for 24 h at 378C and the OD540 was measured using
an ELISA reader. The calculated IC30 value was the lowest drug
concentration that caused a 70% reduction in the OD540 of the
untreated control. The IC30 value was preferred to the minimal
inhibitory concentration (MIC) since the IC30 value is considered to
be more reliable and independent of the inoculum size.15,16
For hyphal induction, semi-synchronized cells (107 cells/mL)
were incubated in Lee’s medium pH 6.8, 5 mM ornithine/10 mM
glucose in PBS, 5 mM N-acetylglucosamine (GlcNAc)/10 mM glucose in PBS or 10% human serum at 378C. To analyse the inhibition
of germ tube formation by drugs (G-IC30 value), 100 mL of drug
For incubation in 2-deoxy-D -glucose-supplemented and glucose-free
buffer, semi-synchronized cells (107 cells/mL) were inoculated into
2 mL Eppendorf tubes containing either PBS, 10 mM glucose in
PBS or 10 mM 2-deoxy-D -glucose in PBS supplemented with or
without ciclopirox (32 or 64 mg/L). Incubation was stopped by
10 000 dilution with glucose-free buffer after 4 h at 378C and the
number of cfu was counted.
For pre-treatment with either hydrogen peroxide (H2O2) or menadione, cells were cultivated in 800 mL of Sabouraud 2% glucose to
exponential phase (5 106 1 107 cells/mL) and divided into
50 mL portions. To each portion, 50 mL of 100 mM menadione in
DMSO or 50 mL of 1 M H2O2 was added. Control cultures contained
the corresponding concentration of DMSO. Cultures were incubated
for 60 min (H2O2) or 90 min (menadione) at 378C, harvested and
washed in PBS pH 7.5. Washed cells (final density 107 cells/mL)
were then inoculated into media supplemented with ciclopirox as
described above.
For depletion of cellular catalase activity 4 106 cells/mL were
pre-incubated in modified Sabouraud glucose for 16 h at 378C
supplemented with 20 or 40 mM of the catalase inhibitor 3-amino1,2,4-triazole. Cells were then washed in PBS and exposed to
ciclopirox as described above.
Cell membrane damage assay
To analyse the effect of various antifungal agents on cell membrane
integrity, potassium concentrations were measured in the supernatant of cultures. Semi-synchronized cells (109 cells/mL) were
washed three times with 20 mM sodium phosphate buffer pH 7.2
and added to 1 mL of buffer containing 16 and 32 mg/L ciclopirox
(final cell number 108 cells/mL). Controls contained no drug. The
maximum possible potassium concentration was measured from the
supernatant of 108 untreated cells boiled for 5 min. The potassium
concentration was obtained by flame photometry using CsCl as an
internal standard and defined concentrations of potassium (0 –2 mM)
in 20 mM sodium phosphate buffer pH 7.2.
664
Antifungal action of ciclopirox
Intracellular ATP concentrations
In order to measure the intracellular ATP concentration, semisynchronized cells (107) were incubated in 5 mM proline/10 mM
glucose/PBS supplemented with ciclopirox at 378C for 30 min. The
cell suspension (1 mL) was diluted with 25 mM Tris – EDTA pH 7.8
and heated at 1008C for 4 min. The suspension was cooled on ice,
centrifuged and the ATP content of the supernatant was
measured. Controls contained no drug. ATP was measured using the
luciferin– luciferase bioluminescence-based ‘ATP Determination
Kit’ (Molecular Probes) according to the manufacturer’s instructions.
Activity of catalase, glucose-6-phosphate-dehydrogenase,
cytochrome c peroxidase, and superoxide dismutase
To measure catalase, glucose-6-phosphate-dehydrogenase (G6PDH), cytochrome c peroxidase, and superoxide dismutase (Sod)
activity, cells were grown in 800 mL of Sabouraud 2% glucose to
exponential phase (5 106 1 107 cells/mL) and divided into
50 mL portions supplemented with ciclopirox and incubated (150
rpm) for 90 min at 378C. Cells were harvested and washed twice
with PBS. For cytochrome c peroxidase activity, cells (2 105)
were incubated in Sabouraud glucose for 24 h, harvested, washed in
PBS resuspended in fresh medium (5 107 cells/mL), and exposed
to ciclopirox for 90 min as described above. Cell pellets were lysed
with a bead mill in 500 mL of PBS twice for 3 min on ice and the
extracts were centrifuged for 1 min at maximum speed in a benchtop
centrifuge.
The protein content of the supernatants was measured17 to give
specific enzyme activities for catalase,18 G6P-DH,19 cytochrome c
peroxidase,20 and Sod.21 In order to discriminate between total Sod
and Mn-Sod activity, we used KCN (6.1 mM) to inhibit any CuZnSod activity.
RNA extraction and microarray analysis
11
For array hybridization, C. albicans MicroArrays were pre-hybridized for 45 min at 428C in pre-hybridization mix [5 sodium
citrate (SSC), 1% sodium dodecyl sulphate (SDS), 1% bovine serum
albumin (BSA)] and washed with water and isopropanol. Hybridization solution (25 mL, 50% formamide/10 SSC/0.2% SDS) was
mixed with 25 mL of the purified Cy3- and Cy5-labelled cDNA mix,
boiled for 3 min and spotted on the MicroArray slide. The array was
covered with a 25 44 mm LifterSlip (Erie Scientific) and incubated
overnight at 428C in a hybridization chamber (Corning). Arrays
were washed in 2 SSC, 1% SDS for 15 min, in 1 SSC, 0.2%
SDS for 8 min, and in 0.1 SSC, 0.2% SDS for 5 min at room temperature with agitation. Slides were dried in a 50 mL Falcon tube by
centrifugation at low speed for 4 min.
Hybridized slides were scanned using an Axon 4000B scanner.
Data were extracted using GenePix 4.1 (Axon). Before the extracted
data were normalized, data were adjusted by background correction
(data transformation). Signals with the same intensity as the local
background were neglected.22,23
This transformation was followed by an intensity-dependent per
spot and per chip normalization (LOWESS). Data analysis was
carried out using GeneSpring 5.0 (Silicon Genetics) from triplicate
(two biological independent experiments and one dye swap)
experiments.
Statistical analysis
_ 3 samples. StanAll data presented are given as the average of n >
dard deviations (SD) are indicated in each case. All experiments
were repeated at least once. Shapiro– Wilk-, F- and Student’s t-test
_ 0.05
were carried out to test statistical significance. A value of P <
was taken as significant.
Results
Antifungal activity of ciclopirox is media dependent
RNA was extracted as described previously. mRNA was translated
into cDNA and labelled as follows. Oligo dT (2 mg) was added to
30 mg of total RNA and brought up to 25 mL with RNase-free water.
After denaturation at 708C for 10 min, the RNA was incubated for
1 min on ice and 30 mL of labelling master mix (1 RT buffer
(Invitrogen), 1 mM DTT, 500 mM dATP, dCTP, dGTP, and 100 mM
dTTP) was added. Cy3 dUTP (3 mL, Amersham) was then added to
one sample and Cy5 dUTP (3 mL, Amersham) to the other sample
and cDNA synthesis was initiated with 2 mL of Superscript II
reverse transcriptase (400 U) (Invitrogen). After 2 h at 428C, 3 mL of
20 mM EDTA was added to stop the reaction and 3 mL of 500 mM
NaOH was added to degrade the RNA for 10 min at 708C. Hydrochloric acid (3 mL, 500 mM) was added to neutralize the reaction.
The labelled cDNA was purified using spin columns (Macherey–
Nagel), dried and resuspended in 20 mL of water.
For transcriptional profiling, we used C. albicans microarrays
(Eurogentec) containing 6039 open reading frames (ORFs) and 27
control genes spotted in duplicate on a glass slide. The C. albicans
MicroArray has been developed in collaboration with the European
Galar Fungail Consortium (www.pasteur.fr/recherche/unites/Galar_
Fungail). The Stanford Genome Technology Center generated the
nucleotide sequence data for C. albicans with funding from NIDCR,
NIH and the Burroughs Wellcome Fund. Information about coding
sequences and proteins was obtained from the CandidaDB database
(www.pasteur.fr/recherche/unites/Galar_Fungail/CandidaDB), which
has been developed by the Galar Fungail European Consortium.
Arrays were designed as described under http://www.pasteur.fr/
recherche/unites/Galar_Fungail/arrays.html.
In order to evaluate the antifungal effects of ciclopirox on
C. albicans, we used IC30 values (lowest drug concentration
which caused 70% reduction in the OD540 of the untreated control), G-IC30 values (lowest drug concentration which caused
70% reduction in the germ tube formation of the untreated control) and a viability test (cfu). Two C. albicans strains (5342 ZN
and CBS 562) were tested in various media (Lee’s medium,
YNB and YNB-4% human serum albumin) to quantify the IC30
values for ciclopirox. The susceptibility of both strains was similar; however, the IC30 values were strongly dependent on the
media used. The IC30 values for both strains were 2 mg/L for
Lee’s medium and between 8 and 16 mg/L for YNB. Addition of
human serum albumin further enhanced this effect (128 mg/L for
5342 ZN and 32 mg/L for CBS 562) suggesting that human
serum albumin may bind to the drug and reduce its activity.
Addition of DMSO, which was used to prepare the ciclopirox
stock solution, did not influence the IC30 values at concentrations up to 2.5%. Final concentrations of DMSO did not
exceed 1% in any experiment indicating that the solvent did not
cause any inhibitory effects.
Ciclopirox inhibits germ tube formation of C. albicans
C. albicans is able to grow in two main morphological forms, as
a spherical yeast or as a long filamentous hyphal cell. In order to
investigate the influence of ciclopirox on growth during the
yeast to hyphal transition, semi-synchronized yeast cells of strain
665
Sigle et al.
5342 ZN were incubated at 378C in Lee’s medium pH 6.8
containing ciclopirox at concentrations ranging from 0.125 to
64 mg/L and the percent inhibition of hyphae formation was
monitored after 4 h of incubation. In parallel, cells were incubated under the same conditions in media containing amphotericin B, flucytosine and clotrimazole. As with amphotericin B,
hyphal formation was inhibited by ciclopirox in a concentrationdependent manner (Figure 1). In contrast, flucytosine and clotrimazole did not inhibit germ tube formation even at the highest
concentrations tested. These results were confirmed for cells
grown in hyphal inducing media containing 5 mM proline/
10 mM glucose, 5 mM ornithine/10 mM glucose and 5 mM
GlcNAc/10 mM glucose (data not shown).
Addition of iron ions to culture media reverses the inhibitory
effect of ciclopirox
Since the effect of hydroxypyridones may be caused by the binding of cellular metal ions,8 we investigated whether the addition
of metal ions to culture media may influence the inhibitory effect
of ciclopirox. First, semi-synchronized cells of strain 5342 ZN
(107 cell/mL) were incubated in 5 mM proline/10 mM glucose/
PBS supplemented with 100 mM Ca2+, Mg2+, Mn2+, Fe2+ or Fe3 +
and the percentage of germ tubes was determined. None of the
metal ions significantly reduced the ability to produce germ tubes
(data not shown). Furthermore, the addition of Ca2+, Mg2+ and
Mn2+ did not influence the G-IC30 of ciclopirox in the same medium; however, the addition of both Fe2+ and Fe3 + (G-IC30 64 mg/
L) clearly reduced the effect of ciclopirox compared with cultures
without the addition of metal ions (G-IC30 8 mg/L) indicating that
these ions possibly interacted with the drug and thus prevented
the inhibition of hyphal formation.
Treatment with ciclopirox does not cause cell membrane
damage
ciclopirox has an effect on the cell membrane of C. albicans, we
tested the cell membrane integrity of strain CBS 562 by measuring the extracellular potassium concentration of drug-treated
cells. Damage of the cell membrane should increase the extracellular concentration of potassium in the medium. Semisynchronized cells of CBS 562 (108 cells/mL) were left untreated
or exposed to 16 and 32 mg/L ciclopirox or 4 and 8 mg/L
amphotericin B in 20 mM sodium phosphate buffer pH 7.2 for
30 and 60 min (Figure 2). The maximum possible potassium
concentration (100% in Figure 2) was measured as
0.60 ± 0.01 mM from boiled untreated cells. Amphotericin B at
both concentrations caused more than 90% release of potassium,
but the viability still exceeded 50% after 60 min. In contrast,
exposure to ciclopirox reduced the cell viability to 20% without
causing any release of potassium into the culture medium compared with untreated cells, indicating that cell membrane damage
was unlikely.
Treatment with ciclopirox does not change intracellular
levels of ATP
It has been postulated that hydroxypyridones inhibit essential cellular processes possibly by blocking the uptake of macromolecule
precursors and essential ions into the fungal cell.4 Such a mode of
action would probably cause a direct or indirect depletion of cellular energy. In order to investigate this hypothesis, we measured
the intracellular level of ATP after cells were exposed to ciclopirox. Semi-synchronized cells (107 cells/mL) of strain 5342 ZN
were incubated in 5 mM proline/10 mM glucose/PBS in the presence and absence of 16, 32, or 64 mg/L ciclopirox and 4 or
8 mg/L amphotericin B for 30 min. Cells were extracted with hot
buffer and the ATP content was measured (Figure 3). Treatment
with amphotericin B significantly reduced the cellular content of
ATP at both concentrations whereas ciclopirox had no effect.
Antifungal drugs such as polyenes and azoles act primarily on
the cell membrane of fungi. In order to investigate whether
Figure 1. Influence of ciclopirox and other antifungals on germ tube formation of C. albicans. Semi-synchronized yeast cells of strain 5342 ZN
(107 cells/mL) were incubated at 378C in Lee’s medium containing ciclopirox, amphotericin B (AMB), flucytosine (5FC) or clotrimazole (CLT) at
concentrations ranging from 0.125 to 64 mg/L. The percentage inhibition of
hyphal formation was monitored after 4 h of incubation. Values represent the
averages of at least four different samples (n = 4–8).
Figure 2. Release of potassium ions from C. albicans strain CBS 562 following 30 min (open bars) and 60 min (grey bars) incubation with ciclopirox
(16 and 32 mg/L) and amphotericin B (4 and 8 mg/L). The maximal release
of potassium ( = 100%) was defined as the amount of potassium released by
boiling untreated cells for 5 min. The viability was 100% at time 0. The viability after 60 min is indicated at the top of each bar as the average ± SD
from three independent experiments. Control, no drug.
666
Antifungal action of ciclopirox
Figure 3. Influence of ciclopirox (grey bars) (16, 32 or 64 mg/L) and amphotericin B (open bars) (4 and 8 mg/L) on the intracellular ATP concentration of
semi-synchronized cells (107 cells/mL) of C. albicans strain 5342 ZN after
30 min of incubation in 5 mM proline/10 mM glucose/PBS. The viability was
100% at time 0. The viability after 30 min is indicated at the top of each bar as
the average ± SD from three independent experiments. Control, no drug.
_ 0.05) different from the ATP content of the control.
*Significantly (P <
Similar results were obtained after prolonged incubations and in
media lacking glucose (data not shown). The ATP concentration
of the culture supernatant was not more than 5% of the total content. Therefore, the antifungal activity of ciclopirox was not
caused by a lack of intracellular ATP.
Oxygen influences the fungicidal effect of ciclopirox
Previous data from our group indicate that ciclopirox causes
increased sensitivity to oxidative stress.11 To investigate the influence of oxygen on the fungicidal activity of ciclopirox, we
pre-cultivated cells of C. albicans strain CBS 562 under reduced
oxygen conditions and then exposed these cells to ciclopirox.
Cells were pre-incubated in modified Sabouraud glucose in batch
cultures (4 106 cells/mL) with and without shaking at 378C for
24 h, then harvested and incubated in 5 mM proline/10 mM glucose/PBS supplemented with 32 or 64 mg/L ciclopirox under
aerobic conditions (Figure 4). The viability of cells in control cultures without drug was similar for cells pre-cultured under
oxygen-rich (with shaking) or reduced oxygen (without shaking)
conditions; however, cells pre-incubated under reduced oxygen
conditions were much more susceptible to ciclopirox compared
with cells grown under oxygen-rich conditions.
In order to clarify to what degree the oxygen content of the
pre-cultures influenced intracellular enzymes involved in oxidative stress, we measured the specific activity of catalase, G6PDH and cytochrome c peroxidase in pre-cultured cells. The
specific activity of catalase was almost twice as high in the
oxygen-rich pre-cultured cells compared with the low oxygen
pre-cultured cells (97.8 ± 16.9 versus 46.0 ± 13.8 U/mL of pro_ 0.05), but the specific activity of G6P-DH
tein; n = 8; P <
_ 0.05) and cyto(145.8 ± 7.8% versus 100 ± 8.1%; n = 2; P <
chrome c peroxidase (0.239 ± 0.015 versus 0.076 ± 0.002
_ 0.05) was also higher.
DA460 nm min 1 mg 1; n = 3; P <
Therefore, poor oxygen supply reduced the specific activity of
certain antioxidant enzymes, which in turn, may influence the
susceptibility of cells to ciclopirox.
Figure 4. Influence of oxygen on the susceptibility of cells to ciclopirox.
C. albicans CBS 562 was pre-cultured in modified Sabouraud glucose medium with and without shaking. Pre-cultured cells were incubated in 5 mM
proline/10 mM glucose/PBS supplemented with 32 or 64 mg/L ciclopirox or
without drug (control) for 4 h (oxygen-rich conditions, open bars; reduced
oxygen conditions, grey bars). The viability for each culture is indicated as
the average ± SD from five independent experiments. The viability for cells
pre-cultured under reduced oxygen conditions and incubated with 64 mg/L
ciclopirox was 0 ± 0.084%.
Addition of 2-deoxy-D -glucose reduces whereas addition
of glucose enhances the effect of ciclopirox
Since oxygen seems to play an essential role in the mode of
action of ciclopirox, we questioned whether metabolic activity is
also necessary for the antifungal activity of this drug.
The metabolic inhibitor 2-deoxy-D -glucose prevents the
import of glucose into fungal cells,24 but neither the lack of
glucose in the incubation buffer nor the addition of 2-deoxy-D glucose influenced the viability of C. albicans CBS 562 cells
(Figure 5). However, cells that were incubated in the absence of
glucose or in the presence of 10 mM 2-deoxy-D -glucose had a
higher viability when exposed to ciclopirox compared with cells
incubated in glucose-containing medium supplemented with
Figure 5. Influence of ciclopirox (32 or 64 mg/L) on the viability of semisynchronized C. albicans CBS 562 (107 cells/mL) in PBS (open bars),
10 mM 2-deoxy-D -glucose in PBS (grey bars) and 10 mM glucose in PBS
(filled bars). The viability was 100% at time 0. The viability after 4 h
incubated is shown as the average ± SD for five independent experiments.
Control, no drug.
667
Sigle et al.
ciclopirox (Figure 5) suggesting that 2-deoxy-D -glucose reduced
and glucose enhanced the effect of the drug.
Pre-incubation of C. albicans cells with hydrogen peroxide
or menadione caused higher tolerance to ciclopirox
The production of reactive oxygen species (ROS) may play an
important role in the mode of action of ciclopirox, since oxygen
and metabolic activity are both required for the drug to be fungicidal. In this case, one would expect that the susceptibility of
fungal cells to this drug was related to the accessibility and
activity of enzymes involved in detoxification of ROS. In order
to modulate the intracellular activity of ROS-detoxifying
enzymes, we exposed exponential growing cells of C. albicans
strain CBS 562 to hydrogen peroxide (H2O2) and menadione for
60 and 90 min. Sublethal concentrations of H2O2 (0.5 and 1 mM)
reduced cell growth and enhanced intracellular catalase activity
11 and 17 times compared with untreated cells (Figure 6a).
Menadione (100 mM) doubled the catalase activity within 90 min
(Figure 6b). Menadione also increased the activity of total Sod
activity by one-third compared with untreated cells (Figure 7).
Since higher concentrations of the inducers were toxic to the
cells, we used 1 mM H2O2 and 100 mM menadione for further
experiments. Since G6P-DH provides essential co-factors for the
detoxification of ROS, we also measured the activity of this
enzyme in cells exposed to H2O2 and menadione. In fact, treatment with H2O2 almost doubled the activity of G6P-DH after 1 h
treatment with 1 mM H2O2 compared with untreated cells
_ 0.05). Similarly, treatment
(188 ± 2% versus 100 ± 6%; n = 2; P <
with 100 mM menadione enhanced the activity of this enzyme
_ 0.05).
(146 ± 18% versus 100 ± 7%; n = 3; P <
When C. albicans cells pre-treated with H2O2 or menadione
were washed and incubated in 5 mM proline/10 mM glucose/PBS
supplemented with ciclopirox, we observed a clear protective
effect in these pre-treated cells compared with control cultures
(Figure 8). Pre-treatment with H2O2 or menadione reduced the
growth, but did not kill the cells. The viability of cells pre-incubated for 60 min with hydrogen peroxide (1 mM) was 114 ± 15%
whereas the viability of control culture without inducer was
186 ± 26%. When cells were pre-incubated for 90 min with
menadione (100 mM), the viability was 131 ± 26% and
176 ± 36% for an untreated pre-culture. Therefore, the induction
of enzymes involved in the detoxification of ROS caused a
higher tolerance to ciclopirox.
Ciclopirox induces intracellular glucose-6-phosphatedehydrogenase, but reduces catalase and does not influence
superoxide dismutase activity
Since higher levels of ROS-detoxifying enzymes had a protective
effect for cells treated with ciclopirox, it may be possible that this
drug may itself cause the production of ROS. This may in turn
cause the production or the induction of the activity of ROSdetoxifying enzymes. To prove this hypothesis, we investigated
the influence of ciclopirox on the activity of G6P-DH,
Figure 6. Induction of intracellular catalase activity with hydrogen peroxide
(H2O2) and menadione. Exponential growing cells of C. albicans strain CBS
562 (5 106 cells/mL) were exposed to H2O2 for 60 min (a) and menadione
for 90 min (b). Catalase activity (U/mL of protein) is shown as open bars and
viability [cfu after incubation compared with cfu at time point 0 (100%)] is
shown as grey bars. Values represent the averages ± SD for three indepen_ 0.05) different from the activity of the
dent experiments. *Significantly (P <
control. Control, no drug.
Figure 7. Induction of intracellular superoxide dismutase (Sod) activity with
menadione. Exponential grown cells of C. albicans strain CBS 562
(5 106 cells/mL) were exposed to 100 mM menadione for 90 min. Total Sod
(open bars) and Mn-Sod (grey bars) activity of 50 mL (250 mL of protein/L)
cell extracts were measured as U/mL of protein. Values represent the
averages ± SD of three samples. Control, no drug.
668
Antifungal action of ciclopirox
Figure 8. Fungicidal effect of ciclopirox (32 and 64 mg/L) on the viability
of exponential grown C. albicans CBS 562 (107 cells/mL) pre-incubated
with 1 mM H2O2 (a) or 100 mM menadione (b) (grey bars) compared
with untreated cultures (open bars). Values represent averages ± SD (n = 3).
_ 0.05) difference between samples. Control, no drug.
*Significant (P <
catalase and Sod in exponential growing cells of C. albicans
strain CBS 562. Treatment with ciclopirox induced the activity of
G6P-DH only at higher concentrations (32 mg/L) (Figure 9a),
reduced the activity of catalase at 16 and 32 mg/L by 50%
(Figure 9b) and had only a minor effect on the Sod activity at
both concentrations (not shown).
Figure 9. Effect of ciclopirox (16 or 32 mg/L) after 90 min incubation on
the viability and activity of (a) glucose-6-phosphate-dehydrogenase and
(b) catalase of exponential growing cells of C. albicans strains CBS 562.
Enzyme activity is shown as open bars, viability compared with time point
0 as grey bars. Values represent averages ± SD (n = 3). Control, no drug.
_ 0.05) different from the activity of the control culture.
*Significantly (P <
Therefore, the fungicidal activity of ciclopirox appears to
be independent of the intracellular activity of catalase in
C. albicans.
Genome-wide transcriptional profiling of ciclopirox-exposed
cells
Inhibition of catalase does not influence cellular viability
and does not increase the fungicidal effect of ciclopirox
As shown above, ciclopirox did not induce, but rather reduced
catalase activity. Therefore, catalase itself may be a target of
ciclopirox and the inhibition of this enzyme may explain the
mode of action of this drug. In order to prove whether
reduced catalase activity may influence cell growth and
viability of C. albicans and to investigate the influence of
ciclopirox on cells with depleted catalase activity, we preincubated cells (CBS 562) with the catalase inhibitor 3-amino1,2,4-triazole and exposed these cells to ciclopirox. As
expected, 3-amino-1,2,4-triazole (20 and 40 mM) reduced the
catalase activity of pre-incubated cells. The catalase activity
was 65 ± 13 U/mL of protein in the untreated control culture,
but only 1.7 ± 0.7 (20 mM) and 1.4 ± 0.7 (40 mM) U/mL of
protein in the cultures treated with 3-amino-1,2,4-triazole;
however, pre-incubation with the catalase inhibitor did not
influence the viability of the cells. Furthermore, lower levels of
catalase did not increase the antifungal activity of ciclopirox.
In order to investigate the transcriptional profiling of drugtreated cells, we grew cells under subinhibitory conditions
(0.6 mg/L) in modified Sabouraud glucose medium11 and isolated
RNA as described previously.11 Control cultures were left
untreated and RNA was isolated at the same time point. mRNA
was translated into cDNA and labelled with Cy3 and Cy5.
Labelled cDNA from drug-treated and control cultures were
used to hybridize microarrays representing PCR fragments of the
complete genome of C. albicans (6039 genes).
Out of the 6039 genes of the C. albicans genome, only 25
were found to be significantly up-regulated ( > 2-fold) and only
21 genes were down-regulated ( > 2-fold) in drug-exposed cells
under subinhibitory conditions. The vast majority of the upregulated genes (15 genes) were involved in iron metabolism
(Figure 10 and Table 1). These included known genes encoding
iron reductases (CFL1), iron permeases and transporters (FTR1,
FTR2 and FTH1) which were previously found up- (FTR1,
FTH1) or down- (FTR2) regulated by semi-quantitative
669
Sigle et al.
encoding the superoxide dismutase Sod4 were up-regulated
(Figure 10).
Down-regulated genes included genes with unknown functions
(9/21), general stress response genes (3/21), cell elongation/
invasive growth genes (2/21), phosphate uptake genes (2/21) and
genes with other functions (4/21). Interestingly, one of the
strongly down-regulated genes was CTA1/CAT1, known to
encode catalase.
Discussion
Ciclopirox acts on yeast and hyphal cells of C. albicans
Figure 10. Known and putative functions of genes up-regulated in cells
exposed to subinhibitory concentrations of ciclopirox (0.6 mg/L).
RT–PCR and northern analysis in ciclopirox-exposed cells,11
confirming the quality of the array data. In addition, we also
found a number of strongly up-regulated genes possibly involved
in iron metabolism (by homology) that have not yet been
described in C. albicans (CFL2, CFL12, FET5, FET32, FET33,
FET34, FRE5, FRE31, FRE32). Furthermore, a number of
genes encoding proteins similar to the GPI-protein Rbt5 (RBT5,
RBT2, IPF12101, CSA1), two genes encoding proteins with
unknown functions, two genes encoding transcriptional factors,
one gene encoding a RNA binding protein, one gene encoding
NADP-glutamate-dehydrogenase and one gene (IPF1218/SOD4)
Hydroxypyridones are widely used topical antifungal drugs
with a broad antimicrobial spectrum including the yeast
C. albicans. In this study, we showed that not only the yeast
growth form of C. albicans, but also germ tube formation is
strongly inhibited by ciclopirox at concentrations higher than
1 mg/L (Figure 1). This was possibly due to a general block of
growth rather than a specific inhibition of the morphological
transition as subinhibitory concentrations of 0.6 mg/L did not
influence hyphal formation.11 Both hyphal and yeast forms are
found in mucosal infections of this fungus and it is assumed
that the hyphal form is essential for invasive growth. Therefore,
inhibition of hyphae formation by ciclopirox may not only stop
proliferation on the epithelial surface, but most importantly
may also prevent penetration of this fungus into deeper tissues.
Being exclusively used as a topical drug, the cutaneous
absorption is known to be very low and ciclopirox is quickly
Table 1. List of up-regulated genes in cells exposed to subinhibitory concentrations of ciclopirox (0.6 mg/L) as identified by genome-wide
transcriptional profiling using microarrays
Systematic
Normalized fold up-regulateda
RBT5
CFL2
IPF13493
IPF12101
RBT2
FRE5
FTR1
FRE31
FET34.3eoc
CSA1
FRE32
FET32
FET5
GDH3
FTH1
FET33
FTR2
IPF7711
CCC2
IPF9315
IPF1218/SOD4
SNM1
IPF7023.3
CFL1
CFL12
19.78
8.98
8.81
7.75
7.17
7.05
6.61
6.61
6.39
5.94
5.75
5.48
5.19
4.09
3.81
3.76
3.13
3.05
2.91
2.70
2.64
2.47
2.47
2.31
2.06
P valueb
0.002
1.6910
3.9610
0.006
2.3910
0.029
1.9610
9.4710
1.9410
1.0110
1.2610
0.004
1.9310
0.047
0.011
1.5510
8.1710
0.002
0.021
0.057
0.048
2.4210
0.052
9.3010
0.024
Known or putative function (reference)
6
7
7
7
7
5
5
5
6
4
5
4
4
repressed by TUP1 protein 5 (homology to CSA1) (4)
ferric reductase (by homology)
unknown function
mycelial surface antigen precursor (by homology)
ferric reductase (by homology) (4)
ferric reductase transmembrane component (by homology)
high affinity iron permease (26)
ferric reductase (by homology)
iron transport multicopper oxidase, 3-prime end (by homology)
mycelial surface antigen (by homology) (19)
ferric reductase (by homology)
cell surface ferroxidase (by homology)
multicopy oxidase (by homology)
NADP-glutamate dehydrogenase (by homology)
iron transporter (34)
cell surface ferroxidase (by homology)
low affinity iron permease (26)
related to Neurospora crassa AP-1-like transcription factor (by homology)
putative copper-transporting ATPase (by homology) (36)
putative CCAAT-binding factor subunit (by homology)
similar to superoxide dismutase (by homology)
RNA binding protein of RNase MRP (by homology)
unknown function, 3-prime end
ferric reductase (12)
similar to ferric reductase Fre2p (by homology)
a
As compared with untreated control.
Student’s t-test.
b
670
Antifungal action of ciclopirox
eliminated from the bloodstream. Not only poor uptake and
rapid elimination but also serum proteins possibly interfering
with antifungal activity may limit the use of this drug to
topical treatment.
Ciclopirox does not damage fungal cell membranes
Despite the fact that hydroxypyridones have been in clinical use
for more than two decades, their mode of action is still not clear.
Early studies suggested that the hydroxypyridone ciclopirox inhibits the energy production of fungal cells consequently blocking
the uptake of components essential for growth.4 S. cerevisiae
cells exposed to another hydroxypyridone, rilopirox, showed a
reduced respiratory activity, which may have been caused by the
reduced uptake or decreased metabolism of glucose or by a direct
attack on the mitochondrial electron transport chain. This may be
explained by the high affinity of hydroxypyridones for metal
ions; rilopirox has been shown to inhibit the activity of NADHubiquinone-oxidoreductase.9 If energy depletion was the principal mode of action, one would expect a slow onset of antifungal
activity; however, our study showed that ciclopirox acts instantly
on proliferating and non-proliferating cells which is in disagreement with the view that energy depletion is the main antifungal
principle. Furthermore, the ATP concentration of cells treated
with ciclopirox did not change, suggesting that a lack of energy
resources as the principal mode of action is unlikely. In contrast
to ciclopirox, treatment with amphotericin B caused reduced
intracellular ATP concentrations possibly by damaging the cellular or mitochondrial membranes. This together with the fact that
cells incubated with ciclopirox do not result in the release of potassium ions into the culture medium strongly suggests that this
drug does not directly damage fungal membranes although modification of the cell membrane has been observed in cells treated
with ciclopirox for longer periods.11 However, this damage is
likely to be due to secondary effects.
It is unlikely that the mode of action of ciclopirox is based on
the cellular uptake of amino acids, potassium- and phosphate
ions as suggested by Iwata & Yamaguchi.4 Starvation of amino
acids and phosphate would probably be reflected in the transcriptional up-regulation of amino acid and phosphate transporters;
however, none of the 28 identified genes encoding phosphate or
amino acid transporters or permeases were found to be up-regulated in cells exposed to subinhibitory concentrations of ciclopirox (data not shown).
Role of oxygen and metabolism
C. albicans cells pre-cultivated in static cultures were found to
be more susceptible to ciclopirox compared with cells pre-cultivated under aerobic conditions suggesting that oxygen plays a
major role in the mode of action of this drug. This may be
linked to the production of reactive oxygen species (ROS)
and/or a reduced cellular detoxification of these molecules. Static cultures of S. cerevisiae have been found to produce more
peroxides when transferred to an oxygen-rich environment.25
This was thought to be due to redox active components, which
were reduced under oxygen-poor conditions. Since the redox status was not changed immediately when cultures were exposed to
oxygen again, molecular oxygen was only partially reduced
and the production of reactive oxygen species was fostered.26,27
Furthermore, proteins that are involved in the detoxification of
ROS, were less induced. For example, cytochrome c peroxidase
activity was low under reduced oxygen conditions, but induced
in oxygen-rich conditions.28 When exposed to pure oxygen,
S. cerevisiae produced significantly more Sod and catalase compared with anaerobic cultures.29 This is in accordance with our
data which show that the activity of enzymes directly or
indirectly involved in the metabolism of ROS such as G6P-DH,
catalase, cytochrome c peroxidase and Sod were reduced in
C. albicans cells grown under oxygen-poor conditions and this
may in turn have caused the higher susceptibility to ciclopirox.
In contrast, cells pre-incubated with hydrogen peroxide or menadione had significantly higher levels of antioxidant enzymes
(G6P-DH, catalase and Sod) and were more tolerant to
ciclopirox.
In addition to oxygen, it seems that an active metabolism is
also required, because both the depletion of glucose in incubation media and the addition of the glucose antagonist 2-deoxyD -glucose strongly reduced the antifungal activity of ciclopirox;
however, it is unlikely that an active metabolism is essential
for the uptake of ciclopirox since it has been shown that ciclopirox enters the cell and accumulates intracellularly in an
ATP-independent manner.6,7
Evidence that ciclopirox acts via the binding of iron ions
Two independent experiments provide strong evidence that
ciclopirox acts principally via the binding of iron. First, the
addition of iron ions (Fe2+ and Fe3 + ) strongly reduced the
inhibitory effect of ciclopirox on germ tube formation. Such an
effect was not seen for other metal ions such as Ca2+, Mg2+ and
Mn2+. Ciclopirox –Fe complexes possibly caused the yellow
coloration regularly seen in cultures supplemented with ciclopirox and iron ions. Secondly, genome-wide transcriptional profiling of cells exposed to ciclopirox clearly reflected that the
cells lacked iron: 60% (15/25) of all strongly (more than 2-fold)
up-regulated genes are involved in iron uptake and metabolism.
These include genes such as CFL1 (iron reductase), FTR1 and
FTH1 (iron permeases and transporters) that were previously
found to be up-regulated by semi-quantitative RT–PCR and
northern analysis in ciclopirox-exposed cells.11 It should be
noted that the high-affinity iron permease gene FTR1 and the
low-affinity iron permease gene FTR2 are highly similar. Therefore, spots on the microarray representing these two genes are
likely to hybridize with either FTR1 or FTR2 probes. Since we
previously showed that FTR1 was up-regulated in cells treated
with ciclopirox, whereas FTR2 was down-regulated,11 we concluded that the signals observed for FTR2 (Table 1) were due to
cross-hybridizations with probes representing FTR1 mRNAs.
In addition, we also found a number of strongly up-regulated
genes possibly involved in iron metabolism (by homology) that
have not yet been described in C. albicans (CFL2, CFL12,
FET5, FET32, FET33, FET34, FRE5, FRE31, FRE32, CCC2).
An orthologue of the C. albicans FRE family has recently been
identified in a screen for genes involved in response to ciclopirox treatment of S. cerevisiae.30 Therefore, transcriptional profiling clearly reflects iron-limiting conditions providing valuable
information regarding the mode of action of ciclopirox. The
observed transcriptional profile of ciclopirox-exposed cells is
unlikely to be a general phenomenon of drug treatment as cells
exposed to azoles exhibit a gene expression pattern which
clearly differs from the data presented here.11,31
671
Sigle et al.
Iron limitation may have multiple effects on the fungal cell.
One likely effect is the reduced activity of proteins that require
iron ions as an essential cofactor. This would include iron–sulphur (Fe/S) proteins such as succinate dehydrogenase and aconitase, haem proteins such as peroxidases, oxidases, cytochrome
P450, and non-haem and non-Fe/S proteins such as dioxygenase,
oxygenase, lipoxygenases and ribonucleotide reductase. The loss
of sufficient activities of all these iron-dependent enzymes may
cause a defined or cumulative or synergic effect resulting in
cell death.
One further possible consequence of iron limitation (caused
by ciclopirox) and oxygen exposure may be an enhanced level
of ROS. This may be due to a higher production of ROS and/or
a reduced detoxification via a blocked activity of antioxidant
enzymes. Since iron is an essential co-factor for the antioxidant
enzyme catalase, it is possible that the activity of this enzyme is
reduced under iron-limited conditions; however, it should also
be noted that high iron levels may itself stimulate or cause the
production of ROS. Therefore, cells need to regulate the concentration of iron since there is a delicate balance between levels
that are essential and those that are toxic.
Our data show that the enzyme activity of two key antioxidant proteins, catalase and Sod, and/or the gene expression
levels of their corresponding genes are altered in ciclopiroxexposed cells. Out of the 6039 genes of the C. albicans genome,
only 25 including IPF1218, which encodes a putative Sod, were
found to be up-regulated ( > 2-fold). However, the total Sod
activity in ciclopirox-treated cells was not altered compared with
control cells suggesting that either other Sods cause the major
Sod activity and/or that post-transcriptional modifications or cofactors are responsible for the reduced activity.
Of the 21 genes which were found to be down-regulated
( > 2-fold) in drug-exposed cells, the catalase gene CAT1
(CTA1)32 was 2.5-fold down-regulated. In addition, the catalase
activity was significantly reduced in drug-exposed cells. Since
catalases belong to the group of iron-dependent enzymes, we
concluded that the reduced activity is due to both a reduction at
the transcriptional level and reduced accessibility to the co-factor
iron. These data confirm our previous observation that the sensitivity of ciclopirox-exposed cells to oxidative stress increased
dramatically.11 However, the reduction in catalase activity is
unlikely to be the principal mode of action which causes cell
death since8 catalase inhibitors did not reduce fungal growth and
did not add to the antifungal activity of ciclopirox (see above)
and18 mutants lacking CAT1 were still viable.32
In summary, this is the first study combining microbiological,
biochemical and genome-wide transcriptional profiling analyses
to elucidate the mode of action of an antifungal drug. The data
obtained in this study indicate that oxygen accessibility and iron
levels are critical parameters in the antifungal activity of ciclopirox possibly by influencing the level of reactive oxygen
species.
Acknowledgements
We wish to thank Donika Kunze, Robert Koch-Institut, Berlin,
Germany, for preparing RNA samples, Florian Wagner, German
Resource Center for Genome Research, Berlin, Germany, for
technical help with scanning microarrays and Caroline Westwater, Medical University of South Carolina, Charleston, SC,
USA, for critical reading of the manuscript. This work has been
supported by the Free University of Berlin and the Robert KochInstitute.
References
1. Dittmar, W. & Lohaus, G. (1973). HOE 296, a new antimycotic
compound with a broad antimicrobial spectrum. Laboratory results.
Arzneimittelforschung 23, 670–4.
2. Gupta, A. K. (2001). Ciclopirox: an overview. International
Journal of Dermatology 40, 305– 10.
3. Dittmar, W., Grau, W., Raether, W. et al. (1981). [Microbiological laboratory studies with ciclopiroxolamine (author’s translation)].
Arzneimittelforschung 31, 1317– 22.
4. Iwata, K. & Yamaguchi, H. (1981). [Studies on the mechanism
of antifungal action of ciclopiroxolamine/Inhibition of transmembrane
transport of amino acid,K+ and phosphate in Candida albicans cells
(author’s translation)]. Arzneimittelforschung 31, 1323– 7.
5. Iwata, K. & Yamaguchi, H. (1980). Mechanism of Action of
Antimycotics. Gustav Fischer Verlag, Stuttgart /New York.
6. Sakurai, K., Sakaguchi, T., Yamaguchi, H. et al. (1978). Mode
of action of 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone ethanolamine salt (Hoe 296). Chemotherapy 24, 68–76.
7. Sakurai, K., Sakaguchi, T., Yamaguchi, H. et al. (1978). Studies
on uptake of 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone ethanolamine salt (Hoe 296) by Candida albicans. Chemotherapy 24,
146–53.
8. Abrams, B. B., Hanel, H. & Hoehler, T. (1991). Ciclopirox
olamine: a hydroxypyridone antifungal agent. Clinics in Dermatology 9,
471–7.
9. Kruse, R., Hengstenberg, W., Hanel, H. et al. (1991). Studies
for the elucidation of the mode of action of the antimycotic
hydroxypyridone compound, rilopirox. Pharmacology 43, 247 –55.
10. Gillum, A. M., Tsay, E. Y. & Kirsch, D. R. (1984). Isolation of the
Candida albicans gene for orotidine-50 -phosphate decarboxylase by
complementation of S. cerevisiae ura3 and E. coli pyrF mutations.
Molecular and General Genetics 198, 179 –82.
11. Niewerth, M., Kunze, D., Seibold, M. et al. (2003). Ciclopirox
olamine treatment affects the expression pattern of Candida albicans
genes encoding virulence factors, iron metabolism proteins, and drug
resistance factors. Antimicrobial Agents and Chemotherapy 47,
1805– 17.
12. Johnson, E. M., Richardson, M. D. & Warnock, D. W. (1983).
Effect of imidazole antifungals on the development of germ tubes by
strains of Candida albicans. Journal of Antimicrobial Chemotherapy 12,
303–16.
13. Evans, E. G., Odds, F. C., Richardson, M. D. et al. (1975).
Optimum conditions for initiation of filamentation in Candida albicans.
Canadian Journal of Microbiology 21, 338– 42.
14. Lee, K. L., Buckley, H. R. & Campbell, C. C. (1975). An amino
acid liquid synthetic medium for the development of mycelial and yeast
forms of Candida albicans. Sabouraudia 13, 148– 53.
15. Hughes, C. E., Bennett, R. L. & Beggs, W. H. (1987). Broth
dilution testing of Candida albicans susceptibility to ketoconazole.
Antimicrobial Agents and Chemotherapy 31, 643 –6.
16. Johnson, E. M., Richardson, M. D. & Warnock, D. W. (1984).
In-vitro resistance to imidazole antifungals in Candida albicans. Journal
of Antimicrobial Chemotherapy 13, 547–58.
17. Smith, P. K., Krohn, R. I., Hermanson, G. T. et al. (1985).
Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76–85.
18. Aebi, H. (1984). Catalase in vitro. Methods in Enzymology 105,
121–6.
19. Löhr, G. W. & Waller, H. D. (1974). Glucose-6-phosphatedehydrogenase. In Methoden der Enzymatischen Analyse
(Bergmeyer, H. U., Ed.) Verlag Chemie, Weinheim.
672
Antifungal action of ciclopirox
20. Valdivia, E., Martinez, J., Ortega, J. M. et al. (1983). Peroxidase
distribution and isoenzyme pattern in different subcellular fractions
from Saccharomyces cerevisiae. Microbios 36, 149 –56.
21. Oberley, L. W. & Spitz, D. R. (1984). Assay of superoxide
dismutase activity in tumor tissue. Methods in Enzymology 105, 457–64.
22. Lashkari, D. A., DeRisi, J. L., McCusker, J. H. et al. (1997).
Yeast microarrays for genome wide parallel genetic and gene
expression analysis. Proceedings of the National Academy of Sciences
USA 94, 13057– 62.
23. Tao, H., Bausch, C., Richmond, C. et al. (1999). Functional
genomics: expression analysis of Escherichia coli growing on minimal
and rich media. Journal of Bacteriology 181, 6425– 40.
24. Shepherd, M. G. & Sullivan, P. A. (1984). The control of
morphogenesis in Candida albicans. Journal of Dental Research 63,
435– 40.
25. Yurkow, E. J. & McKenzie, M. A. (1993). Characterization of
hypoxia-dependent peroxide production in cultures of Saccharomyces
cerevisiae using flow cytometry: a model for ischemic tissue destruction. Cytometry 14, 287– 93.
26. Bast, A., Haenen, G. R. & Doelman, C. J. (1991). Oxidants and
antioxidants: state of the art. American Journal of Medicine 91, 2S–13S.
27. Dawson, T. L., Gores, G. J., Nieminen, A. L. et al. (1993).
Mitochondria as a source of reactive oxygen species during reductive
stress in rat hepatocytes. American Journal of Physiology 264,
C961– 7.
28. Yonetani, T. & Ohnishi, T. (1966). Cytochrome c peroxidase, a
mitochondrial enzyme of yeast. Journal of Biological Chemistry 241,
2983–4.
29. Gregory, E. M., Goscin, S. A. & Fridovich, I. (1974). Superoxide
dismutase and oxygen toxicity in a eukaryote. Journal of Bacteriology
117, 456– 60.
30. Leem, S. H., Park, J. E., Kim, I. S. et al. (2003). The possible
mechanism of action of ciclopirox olamine in the yeast Saccharomyces
cerevisiae. Molecules and Cells 15, 55– 61.
31. Rogers, P. D. & Barker, K. S. (2003). Genome-wide
expression profile analysis reveals coordinately regulated genes
associated with stepwise acquisition of azole resistance in Candida
albicans clinical isolates. Antimicrobial Agents and Chemotherapy
47, 1220– 7.
32. Wysong, D. R., Christin, L., Sugar, A. M. et al. (1998). Cloning
and sequencing of a Candida albicans catalase gene and effects of
disruption of this gene. Infection and Immunity 66, 1953– 61.
673