FEMS Microbiology Letters 185 (2000) 59^63
www.fems-microbiology.org
Phospholipid and sterol analysis of plasma membranes
of azole-resistant Candida albicans strains
Ju«rgen Lo«¥er
a
a;
*, Hermann Einsele a , Holger Hebart a , Ulrike Schumacher b ,
Claudia Hrastnik c , Gu«nther Daum c
Medizinische Klinik, Abteilung II, Labor Prof. Dr. med. H. Einsele, Eberhard Karls Universita«t Tu«bingen, Otfried-Mu«ller-Str. 10, Tu«bingen, Germany
b
Hygieneinstitut, Eberhard Karls Universita«t Tu«bingen, Tu«bingen, Germany
c
Institut fu«r Biochemie und Lebensmittelchemie, Technische Universita«t, Graz, Austria
Received 8 November 1999; received in revised form 4 February 2000 ; accepted 5 February 2000
Abstract
The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was
compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not
exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of
ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma
membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation
of fluconazole thereby providing a mechanism for azole resistance. ß 2000 Federation of European Microbiological Societies. Published
by Elsevier Science B.V. All rights reserved.
Keywords : Phospholipid ; Sterol ; Plasma membrane ; Candida
1. Introduction
Azole antifungals are frequently used for prophylaxis
and treatment of Candida infections in the increasing number of recipients of bone marrow or organ transplants,
patients receiving intensive chemotherapy and in AIDS
patients. The mode of action of these drugs is through
inhibition of a cytochrome P450 enzyme (CYP51) which
catalyzes demethylation of 24-methylenedihydrolanosterol
in Candida albicans. The consequence of such an inhibition
is a reduction in the intracellular level of ergosterol which
results in growth arrest of the yeast.
Azole resistance in di¡erent Candida species (C. albicans, C. tropicalis) has steadily increased in recent times.
However, the mechanisms of resistance are still not fully
understood. Altered activity of sterol 14-demethylase
(CYP51, Erg11p), the target enzyme of azoles, has been
observed in resistant strains of C. albicans [1] presumably
exerting an e¡ect through reduced azole a¤nity as shown
for the amino acid substitutions T315A and G464S in
* Corresponding author. Tel. : +49 (7071) 2987355;
Fax: +49 (7071) 293179.
molecular modeling and mutagenesis studies [2]. Furthermore, studies with C. albicans indicated the central importance in fungistasis of accumulating 14K-methylergosta8,24(28)-dien-3L,K-diol under £uconazole treatment, because mutants defective in sterol C5-desaturase, an enzyme
needed for 6-hydroxylation, are azole-resistant [3]. In addition, overexpression of genes encoding intracellular ef£ux pumps (e.g. CDR genes) might be responsible for
reduced intracellular £uconazole concentrations [4].
A reduced intracellular drug concentration may also be
the consequence of a reduced capability of the drug to
pass the plasma membrane. The basic structural unit of
the plasma membrane is the phospholipid bilayer [5]. Because all phospholipids are amphipathic, hydrophobic interactions between fatty acids create a bilayer of phospholipid molecules whose polar head groups face the
surrounding water. The fatty acyl chains form a continuous hydrophobic interior about 4 nm thick [6]. In addition, biological membranes contain proteins, which bind
to the fatty acyl core of the bilayer or interact with other
proteins [7]. Small molecules can cross the phospholipid
bilayer with little speci¢city. The transport rate of the
molecule is proportional to its hydrophobicity. The lipid
composition of the membrane in£uences the £uidity of the
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 7 1 - 9
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membrane and thus its transport capacity [8]. It is known
that an altered membrane composition a¡ects the membrane permeability.
In order to test the possible in£uence of an altered
plasma membrane structure on the azole susceptibility of
our clinical C. albicans isolates, ¢ve strains were selected
for this analysis, three with known mechanisms of resistance and two with unknown mechanisms of resistance. In
over 30% of the azole-resistant C. albicans strains isolated
from patients with AIDS or hematological malignancies in
our hospital, a speci¢c molecular resistance mechanism
cannot be determined.
2. Materials and methods
2.1. Strains and test conditions
All azole-resistant C. albicans strains (R1^R5) were isolated from AIDS patients receiving repetitive £uconazole
therapy at 100^800 mg day31 for several years. The sensitive strains (S1^S3) were isolated from patients with hematological malignancies. Yeast cells were cultured in RPMI
1640 medium. For antifungal susceptibility testing, yeast
cells were obtained from plate cultures incubated at 37³C
on RPMI 1640 medium with 2% Difco Bacto agar and
were inoculated in 2 ml medium at 5000 cells ml31 . Various doses of antifungal drugs were added to the medium
over 3 days at 37³C. Growth was assessed by cell counts
and colony-forming units ml31 on YEPD consisting of 2%
glucose, 2% Difco Bacto peptone, 1% Difco yeast extract
and 2% Difco Bacto agar. Each test was repeated three
times. Minimum inhibitory concentrations (MIC) were
constant. Resistance of the strains was con¢rmed by Etest (AB Biodisk, Solna, Sweden).
2.2. Plasma membrane preparation
Plasma membrane isolation was performed essentially
as described by Serrano et al. [9] and Monk et al. [10].
In brief, portions of 30 g yeast cells harvested at the late
exponential phase were washed twice with distilled water
and homogenized with glass beads in a Merckenschlager
homogenizer (Braun, Melsungen, Germany) in a bu¡er
containing 50 mM Tris, 2.5 mM EDTA and 1 mM phenylmethylsulfonyl £uoride (PMSF). Immediately after cell
disruption the homogenate was adjusted to pH 7.4 with
2 M Tris. After two rounds of centrifugation at 5000Ug
for 10 min, a crude plasma membrane fraction was sedimented from the supernatant by centrifugation for 1 h at
30 000Ug. This membrane fraction was suspended in
10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM PMSF and
20% glycerol and washed once by centrifugation. The
whole preparation was applied to a discontinuous gradient
made of 1 volume 53.5% and 2 volumes 43.5% sucrose in
1 mM EDTA and 10 mM Tris (pH 7.4), followed by a 5-h
centrifugation at 35 000Ug. Puri¢ed plasma membrane
was recovered at the 43.5/53.5% interface. After threefold dilution with ice-cold water, membranes were sedimented at 80 000Ug for 20 min, and the pellet was resuspended in a small volume of bu¡er.
2.3. Quality control of membrane preparations
The quality of preparations was routinely tested by
SDS^PAGE [11]. To exclude contaminations of plasma
membrane preparations with mitochondria, the activity
of the mitochondrial marker enzyme cytochrome c oxidase
was measured in isolated fractions by standard procedures
[12]. To exclude contaminations with endoplasmic reticulum (ER), the activity of the marker NADPH cytochrome
reductase was measured. Contamination with cytoplasmic
proteins was excluded by analyzing the activity of glyceraldehyde phosphate dehydrogenase by Western blotting.
In all cases, contamination of the plasma membrane was
marginal.
2.4. Lipid analysis
Lipids were analyzed as described earlier [13]. Brie£y,
neutral lipids were separated by thin-layer chromatography using silica gel plates and a solvent containing light
petroleum^diethyl ether^acetic acid (70:30:2, v/v). Phospholipids were separated by two-dimensional thin-layer
chromatography. For development in the ¢rst direction,
chloroform^methanol^25% ammonia (65:35:5, v/v), and
in the second direction, chloroform^acetone^methanol^
acetic acid^water (50:20:10:10:5, v/v) were used.
2.5. Ergosterol analysis
Ergosterol and ergosteryl esters were quanti¢ed after
separation by thin-layer chromatography, by direct densitometry at 275 nm using a Shimadzu CS 930 thin-layer
chromatography scanner. Individual sterols were analyzed
after alkaline hydrolysis of the lipid extract by gas-liquid
chromatography using an Hewlett Packard HP5 capillary
column [14].
2.6. Sensitivity to cycloheximide, sorbitol, sodium chloride
and ethanol
Sabouraud agar with cycloheximide (0.4 g l31 ), YPD
medium (1% yeast extract, 2% peptone, 2% glucose, 0.3
M NaCl) and YPD solid medium were obtained from BD,
Heidelberg, Germany.
Yeast cells from all strains (R1^R5, S1^S3) were precultured at 30³C for 24 h in YPD medium. 10 Wl were transferred onto YPD plates containing 1.2 M sodium chloride,
2 M sorbitol, or 6% ethanol, respectively or onto Sabouraud agar plates containing 0.4 g l31 cycloheximide. Plates
were incubated for 48 h at 30³C.
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61
Table 1
Minimum inhibitory concentrations (Wg ml31 ), growth on YPD medium containing cycloheximide (0.4 g l31 ), sorbitol (2 M), sodium chloride (1.2 M)
or ethanol (6%), and de¢ned mechanisms of azole resistance in azole-resistant (R1^R5) and azole-sensitive (S1^S3) isolates
MIC (Wg ml31 ) £uconazole
MIC (Wg ml31 ) itraconazole
MIC (Wg ml31 ) ketoconazole
MIC (Wg ml31 ) amphotericin B
Growth on YPD plates
containing cycloheximide
Sorbitol
Ethanol
Sodium chloride
De¢ned resistance mechanism
R1
R2
R3
R4
R5
S1
S2
S3
128
32
2
0.25
256
32
8
0.25
256
1
3
0.125
128
0.5
0.25
0.125
256
1
0.03
1
1
0.125
0.06
0.06
2
0.125
0.06
0.125
4
0.125
0.06
0.06
yes
yes
yes
yes
amino acid
substitution
G464S
yes
yes
yes
yes
amino acid
substitution
G464S
yes
yes
yes
yes
overexpression
of e¥ux pump
gene BENr
yes
yes
yes
yes
unknown
no
yes
no
yes
unknown
yes
yes
yes
yes
no
yes
yes
yes
yes
no
yes
yes
yes
yes
no
resistant strains was slightly higher compared to sensitive isolates (5.9^7.5% in resistant isolates, 4.8^5.2% in
sensitive isolates). Cardiolipin was not detectable in isolate
S1.
In all strains analyzed, ergosterol was the predominant
plasma membrane sterol. The ergosterol content varied
between 0.078 mg mg31 protein (in R3) and 0.120 mg
mg31 protein (in S1). As an exception, the plasma membrane of R5 showed an ergosterol content of only 0.02 mg
mg31 protein (20% of R1^R4/S1^S3). No accumulation of
14-methyl fecosterol could be observed.
The extremely low ergosterol to phospholipid ratio of
0.08 (12% of R1^R4/S1^S3) suggests that in this strain the
structure of the plasma membrane is severely altered. All
data are mean values from three independent experiments
with a mean deviation of þ 10%.
Isolate R5 showed a hypersusceptibility to ethanol (6%)
and cycloheximide (0.4 g l31 ). No growth inhibition was
observed with these compounds in R1^R4 and S1^S3. All
isolates grew in the presence of sodium chloride (1.2 M)
and 2 M sorbitol (Table 1).
3. Results
MIC values of all C. albicans isolates (R1^R5, S1^S3)
for £uconazole, itraconazole, ketoconazole and amphotericin B are shown in Table 1.
The phospholipid composition of plasma membranes of
eight C. albicans isolates (R1^R5, and S1^S3) is shown in
Table 2. The major phospholipids in all strains were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. Among individual phospholipids, gradual changes were observed. Amounts of
phosphatidylcholine and phosphatidylethanolamine were
found to be increased in azole-sensitive isolates. The polar
head groups of phosphatidylcholine and phosphatidylethanolamine have no net charge. Phosphatidylinositol and
phosphatidylserine could be detected preferentially in resistant isolates. These phospholipids contain a negatively
charged head group. In addition, R5 showed a phosphatidylcholine:phosphatidylethanolamine ratio which was
lower than in the other resistant strains R1^R4 (0.75 in
R5, 1.8 average in R1^R4). The cardiolipin content of the
Table 2
The phospholipid and ergosterol content of ¢ve azole-resistant (R1^R5) and three azole-sensitive (S1^S3) C. albicans isolates
Lipid
Wild-type
S1
22527 (wild-type)
S2
22512 (wild-type)
S3
Average
wild-type
YO 120
R1
I7
R2
Gr 26
R3
PE 6
R4
I4
R5
Phospholipids (mg mg31 protein)
Phosphatidic acid (%)
Phosphatidylserine (%)
Phosphatidylethanolamine (%)
Phosphatidylcholine (%)
Phosphatidylinositol (%)
Cardiolipin (%)
Dimethylphosph. ethanolamine (%)
Lyso-phospholipids
Other phospholipids
Ergosterol (mg mg31 protein)
Ergosterol/phospholipid
0.240
16.9
21.1
26.2
15.5
7.9
n.det.
n.d.
5.5
4.9
0.120
0.50
0.180
6.7
9.3
25.8
39.9
8.5
7.8
n.d.
n.d.
1.7
0.162
0.90
0.105
12.1
20.8
16.3
31.4
10.9
5.2
n.d.
n.d.
3.2
0.062
0.59
0.175
11.9
17.1
22.8
28.9
11.7
4.3
n.d.
n.s.
3.3
0.115
0.66
0.142
8.9
11.1
14.0
28.8
14.2
7.5
n.d.
3.5
8.1
0.087
0.61
0.116
11.5
18.5
11.9
20.8
12.0
7.1
9.5
0.75
7.7
0.089
0.77
0.108
13.0
20.0
14.4
28.0
12.1
5.9
3.3
0.9
3.9
0.078
0.72
0.164
15.2
28.9
16.2
23.6
8.6
6.4
n.d.
n.d.
0.2
0.106
0.64
0.243
19.1
20.0
23.9
18.1
11.6
6.3
n.d.
n.d.
1.1
0.020
0.08
n.d. : not determined ; n.det. : not detectable ; n.s.: not signi¢cant.
Data are mean values from three independent experiments with a mean deviation of þ 10%.
FEMSLE 9311 15-3-00
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4. Discussion
Azole resistance has become an increasing problem during recent years, partially due to the fact that the underlying mechanisms are not yet fully understood. Changes in
v5,6-desaturase [15] or an increased activity of intracellular e¥ux pumps [16] have been described as mechanisms
of resistance against azoles. Recent data demonstrate that
the G464S amino acid substitution (in R1 and R2) in the
C. albicans sterol 14-demethylase causes £uconazole resistance [17].
In bacteria, decreased accumulation of antibiotics is a
common mechanism of resistance [18]. In fungi, such as C.
albicans, reduced plasma membrane permeability to azoles
has been reported to cause resistance [19]. This has been
found to be associated with changes in the membrane lipid
composition [20]. Recently, in Saccharomyces cerevisiae
[21], it could be demonstrated that an altered phospholipid
and sterol composition of the plasma membrane may
change the membrane barrier function.
In contrast to studies published previously [8], this analysis of the phospholipid and sterol composition of the
plasma membrane was performed with puri¢ed plasma
membrane fractions isolated by ultracentrifugation in
order to obtain a most accurate lipid analysis of this
compartment. Thus, the risk of contaminations with
phospholipids from mitochondrial or ER membranes
was excluded.
The major phospholipids in all strains analyzed in this
study were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. Isolate R5 showed a phosphatidylcholine:phosphatidylethanolamine ratio which was lower than in the other
resistant strains R1^R4.
In addition, the resistant isolate R5 showed only 20% of
the amount of ergosterol detected in R1^R4. In isolates
S1^S3 and R1^R4, a sterol to phospholipid ratio of 0.5^
0.9 was estimated which is in agreement with data reported for the resistant C. albicans Darlington strain
[22]. In contrast, the sterol to phospholipid ratio in the
plasma membrane of R5 was only 0.08, which is only
12% of the sterol to phospholipid ratio of the other
strains. It has been demonstrated in oat and rye shoot
plasma membranes [23] that a reduction of the ergosterol
component of the membrane in£uences the plasma membrane permeability, which may also a¡ect the capacity of
the fungal cell to import azole.
In the present study, isolate R5 with a reduced ergosterol content in the plasma membrane did not grow in the
presence of cycloheximide (0.4 g l31 ), a common protein
biosynthesis inhibitor, or ethanol (6%), causing osmotic
stress. Many studies have reported that changes in the
ergosterol and lipid composition of the plasma membrane
result in a lower tolerance to ethanol [24,25]. Altered
membrane permeability may also lead to a hypersusceptibility to cycloheximide [26]. In contrast, growth was not
inhibited in the presence of sodium chloride and sorbitol.
These results con¢rm the role of the observed change in
the plasma membrane composition to directly a¡ect the
permeability.
Acquired resistance to amphotericin B is reported frequently for Candida lusitaniae. This phenomenon is often
associated with an alteration of membrane lipids and especially a lack of ergosterol [27].
We could previously demonstrate that a lack of ergosterol in the plasma membrane of C. albicans is the cause
of cross-resistance to amphotericin B [15]. In one isolate,
no ergosterol was detectable, leading to complete resistance to amphotericin B (MIC 4 Wg ml31 ), whereas R5
showed a reduced amount of ergosterol in its plasma
membrane, which might be the cause of a weak cross-resistance to amphotericin B (MIC 1 Wg ml31 ).
In conclusion, the decreased phosphatidylcholine:phosphatidylethanolamine ratio in R5 (which was not related
to a known resistance mechanism) combined with a decrease of ergosterol in the plasma membrane might be
responsible for a reduced uptake and thus for a reduced
intracellular accumulation of £uconazole. However, further studies are required to evaluate this possible resistance mechanism of Candida spp. to azole derivatives.
Acknowledgements
This work has been supported by the Deutsche Krebshilfe, Grant 70-2199-Ka1 and the Fortune Program, University of Tu«bingen, Tu«bingen, Germany.
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