Full text of DOI:10.1111/j.1348-0421.2002.tb02702.x

Microbiol. Immunol., 46(5), 317―326, 2002
A Novel Mechanism of Fluconazole Resistance
Associated with Fluconazole Sequestration
in Candida albicans Isolates from
a Myelofibrosis Patient
Kazunori Maebashi*, 1, Michinari Kudoh1, Yayoi Nishiyama1, Koichi Makimura1,
Katsuhisa Uchida1, Takeshi Mori2, and Hideyo Yamaguchi1
1Teikyo University Institute of Medical Mycology, Hachioji, Tokyo 192
―
0395, Japan, and 2Department of Internal Medicine,
Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113
―8421, Japan
Received January 15, 2002. Accepted February 22, 2002
Abstract: A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly iso-
lated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from
TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after
antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate
and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two
resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome
P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of
[3H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the
resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000×
g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultra-
structure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of
numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either
of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the
electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered
in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of
the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affin-
ity of cytochrome P-450.
Key words: Candida albicans, Fluconazole sequestration, Resistance mechanism, Myelofibrosis
Candida albicans is an important opportunistic fungal
pathogen of humans and the major cause of oropharyn-
geal candidiasis (OPC) in AIDS patients (3, 20). The tri-
azole antifungal agent fluconazole has been widely used
to treat patients with OPC. However, the emergence
of C. albicans strains resistant to azole antifungal drugs
because of prolonged fluconazole treatment is now a
recognized problem in AIDS patients with OPC (3, 20).
Several possible mechanisms of resistance to azole
antifungal agents in C. albicans isolates from AIDS
patients have been reported (32). In a great many of
these azole-resistant C. albicans isolates, however, resis-
tance is associated with reduced cellular influx and/or an
increased efflux of azole drugs, especially fluconazole
(21, 29). The increased expression of the multidrug
efflux genes of the ATP-binding cassette family (ABC),
such as CDR genes, and of the major facilitators super-
Abbreviations: ABC, ATP-binding cassette family; CO, carbon
monoxide; CYP450, cytochrome P-450; Erg11p, cytochrome P-
450 dependent sterol 14α-demethylase; IC50, 50% inhibition
concentration; MFS, major facilitators superfamily; NCCLS,
National Committee for Clinical Laboratory Standards; OPC,
oropharyngeal candidiasis; P-120, pellet fraction by centrifugation
at 12,000×g; P-1050, pellet (microsome) fraction by centrifu-
gation at 105,000×g; RT-PCR, reverse transcriptase-polymerase
chain reaction; S-22, supernatant (cell homogenate) fraction by
centrifugation at 2,200×g; S-1050, supernatant (cytosol) fraction
by centrifugation at 105,000×g; YPG broth, broth medium con-
taining 1% yeast extract, 1% Bacto peptone, and 2% glucose.
*Address correspondence to Dr. Kazunori Maebashi, Phar-
maceutical Research Center, Meiji Seika Kaisha Ltd., 760
Morooka-cho, Kouhoku-ku, Yokohama, Kanagawa 222
―8567,
Japan. Fax:＋81―45―541―2359. E-mail: kazunori_maebashi
@meiji.co.jp
317

318
K. MAEBASHI ET AL
family (MFS), such as CaMDR1 gene, has been fre-
quently reported in fluconazole-resistant strains (1, 2, 11,
22, 23, 30). Another common mechanism of resistance
is based on changing the target enzyme of azole drugs,
which is a cytochrome P-450 (CYP450) dependent sterol
14α-demethylase (Erg11p) (28). The alternation of the
ergosterol pathway through a decreased affinity of
CYP450 for azole drugs by mutations in the ERG11
gene that have been identified in several azole-resistant
strains (8, 9, 24, 31), an increased expression of Erg11p
(2, 30), and a defective ∆5,6-sterol desaturase are also
radioactivity: 740 GBq/mmol) was prepared by unil-
abeling with tritium at Amersham Ltd. Unless specified,
other chemicals were purchased from Sigma Chemical
Co., St. Louis, Mo., U.S.A. or Wako Pure Chemical
Industries, Tokyo.
Strains. A series of 10 strains of C. albicans repeat-
edly isolated from blood cultures with a myelofibrosis-
complicated patient were used in this study (Table 1)
(14). All these isolates are thought to be identical by the
analysis of pulsed-field gel electrophoresis of chromo-
somal DNA (15). C. albicans ATCC 10231 was used for
the susceptible control strain and C. albicans TIMM
3165 for the fluconazole-resistant control strain,which
showed an overexpression of CDR mRNA (11). C.
albicans strains were maintained on Sabouraud dex-
trose agar and cultured in YPG broth medium (1% of
yeast extract, 1% of Bacto peptone, and 2% of glucose)
before use.
Susceptibility testing. The MIC of antifungal agents
for C. albicans isolates was determined according to
the National Committee for Clinical Laboratory Stan-
dards (NCCLS) guideline M27-A for broth microdilution
testing (16).
Measurement of sterol 14α-demethylase activity of
growing cells and cell-free extracts. The inhibition of
sterol synthesis by fluconazole was determined by
assessing the incorporation of [14C]acetate (5 µCi, 58
µCi/mmol) and [14C]mevalonate (0.25 µCi, 57 µCi/mmol)
into ergosterol of growing cells and cell-free extracts,
respectively, as described (11).
Sterol analysis. Cells were grown in YPG broth at 37
C for 16 hr, harvested by centrifugation at 1,500×g
for 5 min and washed with distilled water. After the cell
pellet was saponified at 90 C for 1 hr in 50% ethanol
solution containing 15% potassium hydroxide and 0.1%
pyrogallol, nonsaponifiable lipids were extracted three
times with hexane and dried under nitrogen gas. Fol-
lowing silylation at 60 C for 10 min with TMS-HT
(Tokyo Kasei Kogyo Co., Ltd, Tokyo), sterols were ana-
lyzed by gas chromatography-mass spectrometry
(Hewlett Packard, HP-5890/HP-5970). Sterols were
identified by relative retention times of reference, as
described (5, 19, 21), and by mass spectra.
Carbon monoxide difference spectra of CYP450.
Cells were grown semi-anaerobically with stirring to a
logarithmic growth phase at 30 C for 13 hr in YPG
broth containing 4% glucose, harvested by centrifugation
at 1,500×g for 5 min, washed, and suspended in buffer
A consisting of 50 mM potassium phosphate (pH 7.5), 1.2
M sorbitol, 10 mM EDTA, and 10 mM 2-mercaptoethanol.
After incubation at 30 C for 30 min, they were digested
with 15 mg of zymolyase 20T (Seikagaku Kogyo Co.,
Tokyo) per one-gram wet weight of cells at 30 C for 90
responsible for the resistance (4
―
6, 17).
In the past few years, several patients with hemato-
logical disorders are reported to have developed can-
didemia caused by azole-resistant C. albicans, and all
these patients developed resistance following a short
period of exposure to fluconazole (12, 14, 17). This
rapid development of infection caused by azole-resistant
C. albicans is a phenomenon that has not been reported
in AIDS patients (20). One resistant mechanism has
been described in C. albicans isolates from marrow
transplantation patients that became transiently resis-
tant to fluconazole as a result of increased expression of
the CDR gene family (13). On the other hand, two iso-
lates of fluconazole-resistant C. albicans from patients
with leukemia had membrane sterol changes that were
consistent with alternations in a defect of ∆5,6-sterol
desaturase (17). These two isolates were strongly resis-
tant to fluconazole and showed a higher intracellular
accumulation of fluconazole than the susceptible iso-
lates. The same phenomenon was reported in the well-
studied azole-resistant C. albicans strain, Darlington
(1). However, the relationship between fluconazole
resistance and the increased drug accumulation has not
previously been elucidated in these three strains.
The subcellular drug sequestration causing the
decreased drug accumulation in the target site is thought
to be one of the resistant mechanisms in Saccharomyces
cerevisiae (27) and mammalian cell lines (7, 25, 26). In
this paper, we demonstrate the resistance mechanisms of
fluconazole in a series of C. albicans isolates from one
myelofibrosis patient complicated by recurrent can-
didemia. Besides the common azole-resistance mecha-
nisms, we investigated the sequestration mechanism of
fluconazole resistance in C. albicans cells.
Materials and Methods
Materials. Fluconazole (Pfizer Inc., N.J., U.S.A.)
and itraconazole (Janssen Research Foundation, Beerse,
Belgium) were obtained from commercial sources.
Radioisotopes were purchased from Amersham Ltd.,
Buckinghamshire, U.K. [3H]Fluconazole (specific

319
MECHANISM OF FLUCONAZOLE RESISTANCE
min with gentle shaking. Spheroplasts were harvested by
centrifugation at 1,000×g for 5 min, washed with buffer
A in the absence of 2-mercaptoethanol, and resuspended
in 100 mM phosphate buffer (pH 7.5). Spheroplast sus-
pension was homogenized in a Dounce homogenizer
and centrifuged at 1,500 ×g for 5 min to remove debris
and unbroken spheroplasts. The supernatant was cen-
trifuged at 12,000×g for 20 min twice and at 105,000×
g for 90 min to get the microsomal fraction as a pellet.
The pellet was suspended in a 100 mM phosphate buffer
containing 20% (v/v) glycerol and stored at －80 C
until use. Protein concentrations were determined by the
Lowry method (10), and CYP450 concentration was
measured by the method of Omura and Sato (18). After
microsomal suspensions containing 100 nM of CYP450
with 10 µM of fluconazole were incubated at 37 C for 10
min, they were reduced with a sodium hydrosulfite and
bubbled for 30 sec with carbon monoxide (CO). The CO
Dounce homogenizer with 15 strokes. A sample from
the homogenizer was centrifuged twice at 2,200×g for
5 min to remove debris and unbroken spheroplasts, and
the S-22 (cell homogenate) fraction was obtained as the
supernatant. The supernatant was centrifuged twice at
12,000×g for 20 min to obtain the P-120 fraction as a
pellet, and the supernatant was then centrifuged at
105,000×g for 90 min to collect the S-1050 (cytosol)
fraction as the supernatant and to pellet the P-1050
(microsome) fraction. The radioactivity of each fraction
was determined in the Aloka scintillation counter.
Quantification of CDR and CaMDR1 mRNA in C.
albicans isolates. The semi-quantification of CDR and
CaMDR1 mRNA was based on an amplification by
reverse transcriptase-polymerase chain reaction (RT-
PCR), as described previously (11).
Electron microscopy. Cells were grown in YPG broth
at 37 C for 16 hr, harvested by centrifugation at 1,500×
g for 5 min, washed with 0.1 M cacodylate buffer (pH
7.2), and fixed with 2% glutaraldehyde in the same
buffer at 4 C for 2 hr. After being washed with the
same buffer, they were postfixed in 1.5% potassium per-
manganate at 4 C for 18 hr and washed several times
with distilled water. Prestaining with 1% uranyl acetate
(E. Merck Co., Darmstadt, Germany) followed, and the
fixed specimens were dehydrated in an acetone series and
embedded in Quetol 653 (Nissin EM Co., Ltd., Tokyo).
Ultrathin sections were stained with uranyl acetate and
lead citrate, then examined with a Hitachi H-7000 elec-
tron microscope operated at 75 kV. The negative-stain-
ing method was used to examine the fine structure of the
subcellular fractions (P-120 and P-1050). Specimens
were fixed with 0.1 M cacodylate buffer (pH 7.2) con-
taining 1% glutaraldehyde and 0.6 M sorbitol at 4 C for
2 hr and washed with the same buffer. Then one drop of
fixed specimen was placed on formvar-coated grids and
stained with 2% aqueous uranyl acetate. Specimens
were examined with a Hitachi H-7000 electron micro-
scope.
difference spectrum (∆A: 448
―
490 nm) was recorded at
time intervals of up to 30 min with a Beckman UV
spectrophotometer (Beckman Instruments Japan, Tokyo).
The change in absorbance during incubation after CO
treatment is given as a percentage of the value for the
drug-free controls.
Accumulation of [3H]fluconazole by intact cells and
spheroplasts of C. albicans strains. The accumulation of
[3H]fluconazole (100 nM, 14.8 KBq/ml) in intact cells was
measured by the method described (11). For the accu-
mulation of [3H]fluconazole into spheroplasts, cells were
grown in YPG broth at 37 C for 13 hr and prepared as
described above. Spheroplasts were suspended in buffer
B, consisting of 50 mM potassium phosphate buffer (pH
7.5) and 1.2 M sorbitol, to the organism density of 2×108
organisms/ml. At different time intervals, spheroplasts
were filtered with Whatman GF/C filters and washed
with buffer B containing 50 µM unlabeled fluconazole,
and their radioactivity was then counted in an Aloka
scintillation counter.
Intracellular distribution of [3H]fluconazole in sphe-
roplasts of C. albicans strains. Spheroplasts, prepared as
described above, were suspended in buffer B to the
organism density of 1×109 organisms/ml and incubated
at 37 C for 30 min. After [3H]fluconazole (100 nM,
14.8 KBq/ml) was added, spheroplast suspension was
incubated at 37 C for 60 min. Spheroplasts were har-
vested by centrifugation at 1,000×g for 5 min at 4 C and
washed three times with ice-cold buffer B containing 50
µM unlabeled fluconazole. All subsequent procedures
were conducted at 4 C. To prevent the disruption of
organelles and the release of accumulated fluconazole,
spheroplasts were resuspended in 50 mM potassium
phosphate buffer (pH 7.5) containing 0.6 M sorbitol and
50 µM unlabeled fluconazole, then homogenized in a
Results
Susceptibility of C. albicans Isolates to Antifungal Drugs
A series of 10 isolates of C. albicans, from TIMM
3309 to TIMM 3318, were obtained continuously during
a 16-week period from the blood culture of one patient.
As shown in Table 1, the first five isolates from TIMM
3309 to TIMM 3313 collected within about 4 weeks
after the initial antimycotic therapy were susceptible to
all antifungal agents tested. The latter five isolates from
TIMM 3314 to TIMM 3318 collected from 10 to 16
weeks after antimycotic therapy were markedly resistant
to all azole-agents tested. The susceptibilities of the

320
K. MAEBASHI ET AL
five resistant isolates to fluconazole, itraconazole, and
ketoconazole were decreased below 1/500, 1/500, and
1/250, respectively. The degree of azole-resistance in the
five resistant isolates, especially against itraconazole
and ketoconazole, was markedly higher than in the flu-
conazole-resistant control strain, TIMM 3165, which
showed an overexpression of CDR mRNA. However, the
five resistant isolates were susceptible to amphotericin B
and flucytosine.
In contrast, the ergosterol biosynthesis by cell-free
extracts from the two resistant isolates was partly less
susceptible to fluconazole; the IC50s for the resistant iso-
lates were 3.7-fold higher than for the susceptible strain,
TIMM 3313. As shown in Table 2, an analysis of the
sterols by gas chromatography showed that ergosterol
was the main sterol in the membranes of all strains test-
ed. However, no marked accumulation of ergosta-7,22-
dienol or 14α-methyl fecosterol based on ∆5,6-sterol
desaturase defects was observed in the resistant isolates
(data not shown).
Effect of Fluconazole on Sterol Biosynthesis and Sterol
Analysis
The effect of fluconazole on ergosterol biosynthesis by
growing cells and cell-free extracts was compared
between the two susceptible strains, ATCC 10231 and
TIMM 3313, and the two resistant isolates, TIMM 3314
and TIMM 3318. As shown in Table 2, the inhibitory
effect of fluconazole, as represented by the 50% inhibi-
tion concentration (IC50) on sterol synthesis in growing
cells, correlated with their susceptibility to fluconazole.
Effect on the Carbon Oxide Difference Spectra in
CYP450
CYP450 contents of the microsomes obtained from
the cells of ATCC 10231, TIMM 3313, TIMM 3314, and
TIMM 3318 were 0.11, 0.12, 0.10, and 0.14 nmol/mg of
protein, respectively. The relative affinity of CYP450 for
fluconazole was determined by the inhibitory effects on
the CO-CYP450 complex formation during the 30 min
Table 1. In vitro activity of antifungal agents for fluconazole-susceptible and -resistant strains of Candida albicans
Isolated
date
MIC (µg/ml)b)
Organism
Serotypea)
Fluconazole Itraconazole Ketoconazole Amphotericin B 5-Fluorocytosine
ATCC 10231
TIMM 3165
A
A
―
―
0.5
64
0.03
0.5
ꢀ0.016
0.25
0.5
0.5
0.25
0. 5
TIMM 3309
TIMM 3310
TIMM 3311
TIMM 3312
TIMM 3313
TIMM 3314
TIMM 3315
TIMM 3316
TIMM 3317
TIMM 3318
A
A
A
A
A
A
A
A
A
A
’94.08.05c)
’94.08.22
’94.08.27
’94.08.28
’94.08.31
’94.10.18
’94.11.02
’94.11.16
’94.11.28
’94.11.29
0.12
0.12
0.12
0.12
0.12
ꢀ0.016
ꢀ0.016
ꢀ0.016
ꢀ0.016
ꢀ0.016
＞8
＞8
＞8
＞8
＞8
ꢀ0.016
ꢀ0.016
ꢀ0.016
ꢀ0.016
0.5
0.5
0.5
0.5
0.5
1
1
1
1
1
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
ꢀ0.016
＞64
4
4
4
4
4
＞64
＞64
＞64
＞64
a) The serotype was evaluated in an agglutination assay with commercially available polyclonal rabbit antisera (Iatron Laboratories,
Inc., Tokyo).
b) The MIC was determined under NCCLS M27-A guidelines.
c) Antimycotic therapy with fluconazole and amphotericin B was intermittently administered.
Table 2. Effect of fluconazole on ergosterol synthesis, and the relative percentage of
ergosterol among total sterols in fluconazole-susceptible and -resistant strains of Can-
dida albicans isolates
IC50 (µM) of ergosterol synthesis ina):
Relative amount of
ergosterol
Organism
Growing cellsb) Cell-free extracts
(% of total sterol)
ATCC 10231
TIMM 3313
TIMM 3314
TIMM 3318
2.69±0.29
1.02±0.12
＞100
0.072±0.003
0.064±0.004
0.234±0.023
0.237±0.020
92.1
85.8
76.5
76.0
＞100
a) The value is the means±standard deviations of three different experiments.
b)
The IC50 of sterol synthesis by growing cells was determined with cultures
grown in SAAMF broth.

321
MECHANISM OF FLUCONAZOLE RESISTANCE
following the CO treatment. As shown in Fig. 1, the
complex of fluconazole with CYP450 of the resistant iso-
lates was more easily replaced by the CO-CYP450 com-
plex than those of the susceptible strains.
[3H]fluconazole accumulation by intact cells and sphe-
roplasts between the susceptible strains and the resistant
isolates. As shown in Fig. 2, [3H]fluconazole accumu-
lation by the intact cells of the susceptible strains, ATCC
10231 and TIMM 3313, proceeded linearly for 10 min
and continued until 60 min after the onset of incubation.
Unexpectedly, the amount of fluconazole accumulation
by the two resistant isolates, TIMM 3314 and TIMM
3318, was markedly increased; their values were 4.2- to
4.8-fold higher than that accumulated by the susceptible
strain, TIMM 3313, 30 min after the onset of incubation.
The amount of fluconazole accumulation by sphero-
plasts from the susceptible strains and the resistant iso-
late, TIMM 3318, was greater than that accumulated
by intact cells. However, the relative degree of flucona-
zole accumulation by spheroplasts between the two sus-
ceptible strains and the resistant isolate was similar to
those by intact cells; the value by spheroplasts from
TIMM 3318 was 2.9-fold higher than from the suscep-
tible strain, TIMM 3313.
[3H]Fluconazole Accumulation by Intact Cells and
Spheroplasts
To investigate whether a change of permeability and/or
intracellular accumulation of fluconazole is responsible
for the drug resistance, we compared the time courses of
Intracellular Distribution of [3H]Fluconazole by Sphe-
roplasts
In an examination of why the resistant isolates display
fluconazole resistance in spite of the higher levels of
intracellular accumulation of the drug, the intracellular
distribution of [3H]fluconazole by spheroplasts was deter-
mined according to a serial separation by centrifuga-
tion of the S-22 fraction (cell homogenate) prepared
from the susceptible strains and the resistant isolate.
As shown in Fig. 3, the radioactivity in the S-22 fraction
of the susceptible strain, ATCC 10231, was distributed
37% in the P-120 fraction as a pellet by centrifugation at
Fig. 1. Carbon monoxide displacement of 10 µM fluconazole
from the microsomal cytochrome P-450 of fluconazole-susceptible
and -resistant strains of Candida albicans. The difference in
absorbance with the drug at 448 nm and 490 nm is shown as a
percentage of that with drug-free control. The values for each frac-
tion are the means±standard deviations of triplicate experi-
ments.
Fig. 2. [3H]Fluconazole accumulation by intact cells and spheroplasts from fluconazole-susceptible and -resistant strains of Candida albi-
cans. The value at each time point is the mean of triplicate experiments and did not vary more than 15%.

322
K. MAEBASHI ET AL
12,000×g, 7% in the P-1050 fraction (microsome) as a
pellet by centrifugation at 105,000×g, and 33% in the S-
1050 fraction (cytosol) as a supernatant of P-1050 frac-
tion. The percentage of the radioactivity in the S-22 frac-
tion of TIMM 3313 recovered was 41% in P-120 frac-
tion, 7% in the P-1050 fraction, and 32% in the S-1050
fraction. The radioactivity recovered in the S-22 fraction
of TIMM 3318 was 3.7- and 3.8-fold higher than that of
the susceptible strains, ATCC 10231 and TIMM 3313,
and 62% was distributed in the P-120 fraction, 7% in the
P-1050 fraction, and 12% in the S-1050 fraction. In
the resistant isolate, TIMM 3318, most of the intracellular
[3H]fluconazole was distributed to the P-120 fraction
and the rest to the P-1050 fraction in comparison with the
susceptible strains, but the radioactivity in the cytosol
fraction was equal to or slightly greater than that of the
susceptible strains.
Expression of CDR and CaMDR1 mRNA
Fig. 3. Intracellular distribution of [3H]fluconazole in sphero-
plasts by fluconazole-susceptible and -resistant strains of Candida
albicans. The values for each fraction are the means±standard
deviations of six different experiments.
Cdrp and CaMdr1p multidrug efflux transporters have
been identified as drug resistance mediators in C. albi-
cans. In the present study, we used RT-PCR to semi-
quantitatively determine the expression levels of the
Fig. 4. Transmission electron micrographs of fluconazole-susceptible isolate, TIMM 3313 (A), and -resistant isolate, TIMM 3318 (B),
of Candida albicans. N, nucleus; V, vacuole; M, mitochondria; VV, vesicular vacuoles (arrows). Bar: 1 µm.

323
MECHANISM OF FLUCONAZOLE RESISTANCE
Fig. 5. Negative-staining method on electron micrographs of cell fraction of P-120 (A) and P-1050 (B) isolates from fluconazole-resis-
tant Candida albicans TIMM 3318. The arrows indicate vesicular vacuoles in A. The arrow indicates glycogen particles in B. Bar: 100
nm.
CDR family of genes and of the CaMDR1 gene. The
expression level of ACT1 gene was also determined as an
internal standard. We used primers corresponding to the
conserved walker ATP-binding motifs of CDR1 that
should amplify mRNA from all genes of the CDR fam-
ily (11). No difference in the expression levels of CDR
genes were observed between the two susceptible strains,
ATCC 10231 and TIMM 3313, and the two resistant
isolates, TIMM 3314 and TIMM 3318 (data not shown).
The presence of CaMDR1 mRNA was not detected until
30 cycles of PCR amplification in the susceptible strains
or the resistant isolates.
150 to 400 nm) and were observed around a nucleus.
The vesicular vacuoles were not apparent in C. albi-
cans cells such as the susceptible strains, ATCC 10231
and TIMM 3313. Moreover, the subcellular fractions (P-
120 and P-1050) prepared from TIMM 3318 were direct-
ly observed by negative-staining method. As shown in
Fig. 5A, the P-120 fraction was mostly composed of
vesicular vacuoles (diameter, 80 to 200 nm) together
with mitochondria. In contrast, the P-1050 fraction was
composed primarily of glycogen particles with a few
vesicular vacuoles (Fig. 5B).
Discussion
Observation by Electron Microscopy
The ultrastructural appearances of the susceptible
isolate, TIMM 3313, and of the resistant isolate, TIMM
3318, were examined by the use of transmission electron
microscopy. As shown in Fig. 4A, in TIMM 3313 cells,
a nucleus, mitochondria, endoplasmic reticulum, and
vacuoles were visible in cytoplasm; however, a slightly
large number of vacuoles in the cells was observed in
comparison with ATCC 10231 cells and other usual C.
albicans cells (data not shown). As shown in Fig. 4B, the
most notable alteration observed in TIMM 3318 cells was
the appearance of numerous vesicular vacuoles, which
were constructed by a single membrane (diameter of
The increasing incidence of OPC caused by flucona-
zole-resistant C. albicans in AIDS patients has created a
serious problem in the management of these patients
(20). Recently, infections caused by azole-resistant C.
albicans have reportedly occurred in non-AIDS patients
(12, 14, 17). In a Japanese hospital, a series of azole-
resistant C. albicans was isolated from one patient with
myelofibrosis complicated during fluconazole and
amphotericin B therapy (14). The DNA typing of these
10 sequential isolates, which were C. albicans from
TIMM 3309 to TIMM 3318, was almost identical, and,
it was suggested that the latter five isolates might have

324
K. MAEBASHI ET AL
acquired resistance to fluconazole and other azole anti-
fungals (15). In our preliminary study, nucleotide sub-
stitutions of the ERG11 gene from 330th to 1654th in
TIMM 3313, TIMM 3314, and TIMM 3318 were iden-
tical in all of the 11 positions referred to the published
sequence of the ERG11 gene (GenBank accession no.
X13296) (data not shown). One more nucleotide sub-
stitution was also observed in TIMM 3318. This result
supports the three TIMM isolates having a high degree of
relatedness, and TIMM 3314 and TIMM 3318 could
acquire fluconazole resistant with development from
one original strain. In this paper we therefore attempted
to elucidate the fluconazole resistance mechanisms by
using the series of C. albicans isolates, especially TIMM
3313 as a susceptible isolate just before acquiring resis-
tance, TIMM 3314 and TIMM 3318 as resistant iso-
lates, and C. albicans ATCC 10231 as the susceptible
control strain.
The target molecule of the antifungal action of flu-
conazole and other azole antifungals is known to be
CYP450 dependent sterol 14α-demethylase encoded by
ERG11, a key enzyme on the fungal ergosterol synthet-
ic pathway (28). Compared with the fluconazole-sus-
ceptible strain, TIMM 3313, the two resistant isolates
tested showed being partly less susceptible to fluconazole
on ergosterol synthesis by cell-free extracts caused by the
reduced affinity of CYP450 for fluconazole rather than by
an overproduction of this enzyme. However, the IC50 val-
ues of fluconazole against ergosterol synthesis by grow-
ing cells from the two resistant isolates were markedly
higher than the value for the susceptible strain, TIMM
3313. It appears quite likely, therefore, that besides the
decreased CYP450 affinity, some other biological mech-
anism(s) is also responsible for the fluconazole resistance
in the resistant isolates tested.
The most common mechanism of fluconazole resis-
tance in C. albicans has been reported to be a reduced
intracellular accumulation of the drug as a result of
reduced permeability and/or increased efflux (21, 29).
Previous studies have documented that the efflux of
azole drugs from fungal cells is due to energy-dependent
pumps, and that the increased expression of the CDR
genes (specifically CDR1 and CDR2) and of the
CaMDR1 gene is associated with the development of
resistance (1, 2, 11, 22, 23, 30). In this study, the resis-
tant isolates tested were highly resistant to fluconazole,
despite the accumulation of high intracellular levels of
fluconazole. The same phenomenon was reported in
C. albicans Darlington (1, 4, 5) and two C. albicans
isolates from two patients with leukemia (17). The flu-
conazole-resistant mechanisms of Darlington and these
two strains are believed to be due to a partial inhibition of
∆5,6-sterol desaturase, ultimately synthesizing a meta-
bolic intermediate sterol in place of ergosterol (4, 5,
17). However, in the two resistant isolates tested, the
major sterol in the membranes was ergosterol. These
results rule out the possibility that a defect in ∆5,6-sterol
desaturase contributes to fluconazole resistance. The
resistant mechanism for the resistant isolates tested can-
not be completely explained by the common mecha-
nisms. We therefore investigated the intracellular dis-
tribution of fluconazole between the susceptible strains
and the resistant isolate. In the resistant isolate, TIMM
3318, most of the accumulated fluconazole in sphero-
plasts was distributed in the P-120 fraction, but the level
in the S-1050 (cytosol) fraction was almost equal to the
susceptible strains. These results suggest that the specific
sequestration of fluconazole in the P-120 fraction might
be an important part of the resistance mechanism.
In observation of the thin-sectioned cells, the most
notable ultrastructural finding in the resistant isolate,
TIMM 3318, was the appearance of numerous small
vesicular structures. If it is based on morphological
character, it is difficult to distinguish the vesicular struc-
tures observed in TIMM 3318 cells from normal vacuole.
However, the size and number of the vesicular structures
were obviously different from normal vacuole. To our
knowledge, no other report has been made of such struc-
tures induced in usual C. albicans cells, not any in
another fluconazole-resistant C. albicans, which has not
previously been elucidated morphologically. Most vesic-
ular vacuoles were observed in the P-120 fraction on
electron microscopy by the negative-staining method. In
some mammalian cell lines, drug resistance is induced by
the reduction of cytoplasmic drug levels through the
sequestration of drugs into a vesicular cytoplasmic com-
partment (7, 25, 26). The vacuolar sequestration mech-
anism also plays a significant role in the detoxification of
cadmium in S. cerevisiae through the yeast cadmium
factor encoded by YCF1 gene, which belongs to the
vacuolar glutathione S-conjugate pump of the ABC
superfamily (27). This evidence supports one possible
explanation that fluconazole resistance in the resistant iso-
lates tested might be caused by the vesicular seques-
tration of fluconazole in cytoplasm. In our preliminary
study, the combination of verapamil, carbonylcyanide-m-
chlorophenylhydrazone, rhodamine 6G, or indomethacin
with fluconazole showed synergetic effects against the
two resistant isolates, TIMM 3314 and TIMM 3318.
However, no effects of the four compounds on flucona-
zole susceptibility were noted in both the susceptible
strains and the CDR-expressing resistant control strain,
TIMM 3165. Furthermore, the three of four compounds,
except for indomethacin (not tested), had no affect on flu-
conazole intracellular accumulation in all strains tested
(data not shown). One possible explanation for these

325
MECHANISM OF FLUCONAZOLE RESISTANCE
10) Lowry, O.H., Resebrough, N.J., Farr, A.L., and Randall,
R.J. 1951. Protein measurement with Folin phenol reagent.
results is that the four compounds may interact with the
intracellular distribution of fluconazole in cytoplasm,
and fluconazole may be incorporated in vesicular vac-
uoles as a free form rather than as a metabolite.
Our data show that a novel mechanism of fluconazole
resistance, which is rapidly and transiently developed in
a myelofibrosis patient, might occur by the reduction of
cytoplasmic drug accumulation based on the vesicular
fluconazole sequestration besides a decreased affinity
of the drug for CYP450. It would therefore be of inter-
est in further investigations to examine the direct incor-
poration of fluconazole into vesicular vacuoles.
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mura, K., Niimi, K., Piper, R.J., Uchida, K., Arisawa, M.,
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