MYCOSES
ACCEPTED: JANUARY 8, 1991
34, 75-83 (1991)
Polyene resistance in ergosterol producing strains of Candida albicans Polyen-Resistenz bei Ergosterin-produzierenden Candida albicansStammen M. Christine Broughton, M. Bard and N. D. Lees Key words. Candida albicans, polyene resistance, amphotericin B resistance. Schliisselworter. Candida albicans, pol yen-Resistenz, Amphotericin B-Resistenz.
Summary. Following nitrous acid mutagenesis, one nystatin- (nyl) and two amphotericin B (AB)resistant (abl and ab2) mutants of Candida albicans were isolated and characterized. The three mutants plus a previously described cytochrome P450deficient mutant (D10) of this organism were analyzed for polyene cross resistance. Cross resistance was noted for nyl and D10 but not for abl and ab2. Sterol analysis indicated that nyl was a A8-A7 isomerase mutant while abl and ab2 showed wild type sterol profiles. Fatty acid analysis showed no significant differences for abl, ab2, and nyl compared to wild type while D 10 showed more pronounced differences. AB- and Triton X-1 00-induced potassium leakage studies indicated that abl and ab2 are resistant to low AB levels and nyl is resistant to higher AB levels. In contrast, a b l and ab2 were more resistant to detergent-induced potassium leakage than the wild type or mutants nyl and DlO. Significant differences in growth rate, ethanol sensitivity, and response to Tergitol were also noted among the resistant strains. The data indicate a different mechanism of action for the two polyenes and indicate a resistance mechanism for abl and ab2 based on subtle alterations of membrane structure rather than sterol substitution.
Department of Biology, Indiana University - Purdue University at Indianapolis, Indiana, USA. Correspondence: Professor Dr Norman D. Lees, Department of Biology, Indiana University- Purdue University, 1125 East 38th Street, Indianapolis, IN 46205, USA.
Zusammenfassung. Im AnschluR an eine Mu-
tagenese mit salpetriger Saure wurden Mutanten von Candida albicans isoliert und charakterisiert, eine Nystatin- (nyl) resistente und zwei Amphotericin B (AB)-resistente (abl und ab2). Die drei Mutanten und eine andere, Zytochrom P450-defizitare Mutante (D10) dieses Organismus, welche fruher beschrieben wurde, wurden auf Polyen-Kreuzresistenz analysiert. Kreuzresistenz wurde fur ny 1 und D10 festgestellt, aber nicht fur abl und ab2. Eine Sterinanalyse ergab, daI3 ny 1 eine A8-A7-IsomeraseMutante war, wahrend abl und ab2 Wildtyp-Sterinprofile aufwiesen. Eine Fettsaurenanalyse zeigte keine signifikanten Unterschiede fur abl, ab2 und nyl im Vergleich zu Wildtypen. D10 hingegen wies ausgepragtere Unterschiede auf. Untersuchungen der Kaliumdurchlassigkeit, induziert durch ABund Triton X-100, ergaben, daR abl und ab2 gegen ein niedriges, nyl aber gegen ein hoheres AB-Niveau resistent sind. Im Gegensatz d a m hatten abl und ab2 eine hohere Resistenz gegen eine durch Detergenzien induzierte Kaliumdurchlassigkeit als die Wildtypen oder Mutanten nyl und D10. Signifikante Unterschiede in der Wachstumsrate, Athylalkohol-Sensitivitat und Reaktion auf Tergitol wurden unter den resistenten Stammen ebenfalls festgestellt. Die Daten weisen auf einen unterschiedlichen Wirkungsmechanismus fur die beiden Polyene hin sowie auf einen Resistenzmechanismus fur abl und ab2, welcher eher auf einer subtilen Anderung der Membranstruktur als auf einer SterinSubsti tution beruht.
76
M.C . BROUGHTON ET AL.
Introduction
Methods
Amphotericin B (AB) is an effective antifungal agent that has an extended history of successful use in systemic fungal infections. There are, however, a number of serious side effects [ 13 associated with this drug which have limited its usefulness and led to the development of alternative ways in which to administer the drug [2] and to the development of new antifungal agents [3]. Although resistance to AB has not been frequently reported for clinical isolates, recently the appearance of low level AB resistance in a variety of pathogenic yeasts including C. albicans has been documented in severely immunocompromised patients [4]. In this study [4], as in past studies [5, 61, a thorough analysis of the resistance mechanism was not undertaken. These limited clinical studies, plus more extensive studies of laboratory isolates of ABresistant strains, have resulted in several possible mechanism of AB resistance including cell wall exclusion [7], increased ergosterol content [6], and reduced ergosterol content [8-101. The latter mechanism is most consistent with the extensive data collected using AB, and other polyenes including nystatin, which indicate a relationship between drug affinity for ergosterol, the major fungal membrane sterol, and antifungal activity [ 13. This relationship has been employed in experiments in which ergosterol biosynthetic mutants have been isolated in fungi, including Candida albicans [l 11 and Saccharomyces cerevisiae [9, 101, based on resistance to polyenes. There have been a few reports of low level AB resistance, from clinical and laboratory studies, in which the resistance mechanism seems to be related to factors other than ergosterol content [ 1, 71. Most of these studies go only as far as determining the minimal inhibitory concentrations of AB and performing a simple analysis of cellular sterol content. Resistance to low levels of AB is of great clinical significance since resistance levels as low as 0.8 pg ml-I have been shown to result in patient fatality [4]. The mechanism associated with and the corresponding physiological characteristics of such mutants should be of importance in drug strategies to combat future resistance which seems likely based on the increase in infections with C. albicans [4]. In this study we will describe the characteristics of several polyene resistant mutants of C. albicans, some of which have wild type sterol profiles.
Fungi Nystatin- and AB-resistant mutants were derived from C. albicans strain ATCC 44829. C. albicans strain D10 (ATCC 38247) a nystatin-resistant, cytochrome P450-deficient mutant, has been previously described [ 11, 121. Strain DlOR is a wild type revertant of strain D10 isolated as a fast growing colony in a lawn of D10 on a complete medium without nystatin [12].
Media and growth conditions Cells were grown in 1% Difco yeast extract, 2% Difco peptone, and 2% glucose (YPD). 2% ethanol was used to replace glucose (YPE) or added in addition to glucose (YPDE). The non-ionic detergent, Tergitol NP-40 (Sigma), was added at a concentration of 1.5%. Plates were made by adding 2% agar. Strain D10 was maintained on YPD plates supplemented with 35 pg ml-' nystatin. AB-resistant mutants a b l and ab2 were maintained on YPD plates supplemented with 3 and 1 pg ml-' of AB, respectively. All growth was performed at 30 "C.
An tifungal agen ts Nystatin and AB were obtained from Sigma. Nystatin stocks were prepared at 4 mg ml-I in dimethylformamide and stored a t - 20 "C. AB stocks were prepared at 5 mg ml-I in dimethylformamide and stored at - 20 "C.
Mutagenesis Nitrous acid mutagenesis was performed according to the procedure of Kakar & Magee [ 131. Samples of YPD cultures were collected by centrifugation, washed and resuspended in 0.1 mol sodium acetate buffer, pH 4.5, containing 30 mmol (abl and ab2) or 60 mmol (nyl) sodium nitrite. After 5 min (abl and ab2) or 60 min (nyl) of incubation, the cells were collected by centrifugation and resuspended in 0.1 mol phosphate buffer, pH 7.5, to stop the mutagenesis. The suspensions were transferred to sterile, plastic petri dishes and exposed to short wave ultraviolet irradiation for 15 sec (abl and ab2) or 45 sec (nyl) to promote chromosomal crossing over. For the isolation of AB-resistant mutants, rnutagenized cells were grown overnight in YPD supplemented with 6 pg ml-' AB. Cells from the overnight growth were then plated on YPD supplemented with the same concentration of AB. For the isolation of nystatin-resistant mutants, mutagenized cells were plated directly on YPD supplemented with 5 pg ml-' nystatin. mycoses 34, 75-83 ( 1991)
POLYENERESISTANCE
Minim urn Inhibitory Concentration (MIC) For the determination of MIC on a solid medium, overnight YPD broth cultures were diluted in 0.9% saline to lo3 cells/ml. 0.1 ml of the dilution was spread on a YPD plate supplemented with AB or nystatin. MIC was defined as the lowest concentration at which no growth occurred after 48 h of incubation. For the determination of MIC in a liquid medium, cells from overnight YPD broth cultures were used to inoculate 40 ml of YPD broth supplemented with AB or nystatin. Initial turbidities were 8-15 Klett units as determined using a Klett Summerson colorimeter with a no. 66 red filter. MIC was defined as the lowest concentration at which no increase in turbidity was noted after 24 h of incubation.
Sterol analysis Quantitative sterol analysis was determined for exponential phase cells by inoculating YPD broth with cells from an overnight YPD plate and growing to early exponential phase (20-60 Klett units). 200 ml samples were removed for sterol analysis and 40 ml samples were transferred to tared centrifuge tubes for dry weight determinations. For stationary phase cultures, YPD broth cultures were grown for 48 h. 25 ml samples were removed for sterol analysis and 10 ml samples were transferred to tared centrifuge tubes for dry weight determinations. Dry weights were determined by drying the cell pellets for 48 h at 62 "C and reweighing the tared tubes. Samples for sterol analysis were collected by centrifugation and cells resuspended in 40% alcoholic KOH (25 g K O H dissolved in 36 ml water, brought to 100 ml total volume with 95% ethanol) for saponification. The suspension was transferred to a round bottom flask, a boiling chip added and a reflux tube attached, before being placed in an 8590°C water bath for 2 h. After 1 h, 1 ml of 95% ethanol was added to replace volume lost by evaporation. The flask was removed from the bath and allowed to cool. Water (4 ml) and heptane (10 ml) were added and the mixture vigorously shaken for 3 min. The flask was stored at room temperature for 15 min to allow separation of the organic and aqueous phases. The organic phase was removed for sterol analysis. Sterol analysis was performed on an H P 5710A gas chromatograph using an SE-30 six foot glass column at 240 "C. The carrier gas was nitrogen at 60 ml/min. Five pl of the heptane phase was used for each analysis. Peak retention times and areas were determined with an H P 3392A integrator. Sterol concentrations were determined using ergosterol (0.1 mg ml-' heptane; Sigma) and lanosterol rnycoses 34, 75-83 (199 1)
IN
CANDIDA ALBICANS
77
(0.1 mg ml-' heptane; Sigma) standards' peak areas as references.
Fatty acid analysis Fatty acid analysis was performed according to the procedure of Miller & Berger [ 141. A loopful of cells from an overnight YPD plate was used to coat the bottom of a screw cap glass tube. One ml of saponification reagent (45 g NaOH, 50 ml methanol, and 150 ml water) was added and the tube vortexed. The tube was incubated at 100°C for 5 min, vortexed, and reincubated at 100 "C for 25 min. After cooling in a water bath, 2 ml of methylating reagent (325 m l 6 N HCl and 275 ml methanol) was added and the mixture vortexed prior to incubation at 80 "C for 10 min. 1.25 ml of extraction reagent (1:l ratio of hexane and methyl-t-butyl-ether) was added and the contents of the tube mixed by inversion. Upon phase separation, the bottom aqueous phase was removed. 3 ml of wash reagent (10.8 g NaOH in 900 ml water) was added to the remaining organic phase to remove the fatty acids and the tube was inverted several times for 5 min followed by the addition of 2-3 drops of saturated NaCl to aid phase separation. Approximately 2/3 of the upper, organic phase was removed for fatty acid analysis. Fatty acid analysis was performed using 2 pl samples on an H P 5890GC gas chromatograph using a 25 m x 0.2 mm methyl phenyl silicone fused silica capillary column at 250°C. The carrier gas was nitrogen at 30 ml/min. Fatty acids were identified by relative retention times compared to fatty acid standards and quantitated as yo total fatty acids.
Potassium leakage YPD broth was inoculated from an overnight YPD plate to a concentration of 8-15 Klett units and grown to exponential phase. Cells were prepared, treated, and assayed for potassium leakage as dscribed by Bard et al. [ 151. Cells were collected by centrifugation and washed twice and resuspended in 10 mmol Tris, 0.9% NaCl buffer, pH 7.0, to a final concentration of 3 x lo7 cells/ml. Samples (treated and untreated) were incubated at 30 "C on a shaking water bath a t 200 rpm for 30 min. Cells were removed by centrifugation and the supernatant fluids were assayed on an IL 952 atomic absorption spectrophotometer to determine potassium concentration. Total cellular potassium was determined by boiling a sample for 10-15 min to lyse the cells. One tailed t-tests were performed to determine the statistical significance between critical data points. Probabilities greater than 0.05 were not considered significant.
78
M. C . BROUGHTON
ET AL.
Mean generation time (mgt)
Table 2.
Cells from overnight cultures grown in YPD broth were collected by centrifugation and resuspended at a concentration of 8-15 Klett units in a 250 ml nephelometer flask containing 20 ml of the medium to be tested. Cells were incubated at 30 "C and turbidity monitored at appropriate intervals. The mgt values were calculated during exponential growth phase.
Results Nitrous acids mutagenesis of C. albicans strain 44829 followed by UV irradiation resulted in the isolation of one nystatin-resistant mutant (nyl) and four AB-resistant mutants (abl, ab2, ab3, and ab4). Initial characterization of resistance levels indicated that abl was more resistant (8 times that of wild type) than the other three isolates (3 times that of wild type). Thus, abl and one of the lesser resistant strains, ab2, were chosen for further characterization. In addition, strain D10 of C. albicans, a nystatin-resistant, cytochrome P450-deficient mutant [ l l , 121 and its wild type revertant, strain DlOR, were included in the characterization since this particular mutation represents one of only two verified steps in ergosterol biosynthesis for which there are blocks in this organism. The results of MIC determinations on a solid medium are shown in Table 1. The level of nystatin resistance of strain ny 1 was significantly higher than that seen in the wild type but far below that seen for strain D10. Both of these mutants showed cross resistance to AB. The AB-resistant strains, a b l and ab2, showed 8 and 3 times, respectively, the level of AB resistance seen in the wild type. Both strains showed minimal cross resistance to nystatin. The MIC data in a liquid medium shown in Table 2 indicate a similar but more pronounced pattern of AB resistance. Nystatin resistance levels for Table 1. Minimum inhibitory concentrations*of polyene antibiotics for Candida albicans strains grown on solid media for 48 hours Antibiotic
44829 nyl
Nystatin 3.5b (Pip ml-9 Amphotericin B 2.0 (pg ml-')
ab2
abl
D10
DlOR
25.0
5.0
5.0
75.0
3.0
5.0
6.0
16.0
30.0
1.5
' lowest concentration of antibiotic necessary to completely inhibit growth data represented as means of 2 or 3 experiments. Variation among experiments was 1 pg ml-' or less for all entries
Minimum inhibitory concentrationsaof polyene antibiotics for Candida albicans strains grown in liquid media for 24 hours
I Antibiotic
~~
DlOR
44829 nyl
ab2
abl
D10
10.0
40.0
10.0
10.0
150.0 5.0
Amphotericin B 1.0 ( M ml-9
25.0
3.0
5.0
150.0 1.0
I
Nystatin
(Md-9 I
a
lowest concentration of antibiotic necessary to completely inhibit growth data represented as means of 2 or 3 experiments. Variation among experiments was 1 pg ml-1 or less for all entries
all strains were uniformly higher. Strain D10 showed very high resistance while the level for strain nyl was four times that of its wild type. Both of these strains showed significantly greater cross resistance to AB compared to the solid medium MIC results. With the exceptions of strains D10 and nyl, AB resistance levels were lower in the liquid medium compared to the solid medium. In addition, ab l and ab2 showed no cross resistance to nystatin. Sterol analysis of exponential phase cells, shown in Table 3, indicated a typical, wild type sterol profile for strains 44829 and DlOR, with ergosterol present as the dominant sterol and low levels of lanosterol [5, 11, 121. Previously, more extensive analysis (gas chromatography-mass spectral analysis) of strain DlOR [12] indicated the presence of two additional minor sterols that were identified as ergosta-dien-3P-01s. Strain nyl showed no detectable egosterol. Instead, it accumulated a mixture of delta-8 sterols, that have since been identified (Barbuch et al., unpublished) as ergosta-5,8,22-trien-3P01, ergosta-8-en-3P-01, and fecosterol. This sterol composition is consistent with that reported for the erg2 mutant of S. cerevisiae [9,101 and the E4 mutant of C. albicans [l l ]. Strain D10 accumulated the five methyl sterols previously reported [ 121. Strains abl and ab2 produced sterol profiles essentially identical to that of the wild type strain. Total sterol content was 3 0 4 0 % higher for nyl compared to the wild types and the AB-resistant strains while strain D10 produced considerably less sterol. In general, sterol analysis of stationary phase cells (Table 4) indicated uniformly higher sterol content and profiles similar to those noted in exponential phase cells for all strains. Exceptions, were a significant increase in ergosta-5,8,22-trien-3P-ol for ny 1 and an elevated ergosterol level in abl. Since some nystatin-resistant mutants of S. cerevisiae showed altered fatty acid composition [ 1 13, this feature of the strains was examined. The data in Table 5 indicate that strains nyl, ab2, and abl showed no significant difference in total cellular fatmycoses 34, 75-83 (1991)
POLYENE RESISTANCE IN GNDIDA ALBICANS
I Table 3.
79
Sterol content (pg sterol/mg dry weight) of exponential phase cultures of Candida albicans strains Strains
Sterol
44829
nYl
ab2
abl
DlOR
4.12f0.12
4.03f0.66
3.97k0.72
D10
I
1.45ak0.13b
ergosta-5,8, 22-triene-3P-01 3.81 k 0.38
ergosterol
0.60f0.16
14-methylfecosterol 4.32 a 0.27
ergosta-8-en-3P-01 & fecosterol
0.42 f 0.13
obtusifoliol 0.16 f 0.02
lanosterol
0.27k0.09
0.34k0.05
0.26k0.02
24-meth ylene-24, 25-dihydrolanosterol
0.42 f 0.17 0.03 f 0.02
25-dih ydrolanosterol Total sterols a
0.37f0.15
3.97f0.32
5.77f0.38
4.29f0.20
4.27k0.70
4.23k0.74
1.84k0.52
all means calculated from at least three experiments standard deviation
I Table 4.
Sterol content (pg sterol/mg dry weight) of stationary phase cultures of Candida albicans strains Strains
Sterol
"Y 1
44829
ab2
abl
DlOR
D10
I
ergosta-5,8, 22-triene-3P-ol ergosterol
4.12' k 0.45b 5.84 f 0.38
5.41
f
0.17
6.29
f
0.19
6.21
f
0.33 0.46 f 0.31
14-methylfecosterol 4.77
ergosta-8-en-3P-01 & fecosterol
f
0.69
obtusifoliol lanosterol
1.01 0.40 f 0.07
0.46 f 0.07
0.74 f 0.01
0.08 f 0.06
24-methy he-24, 25-dihydrolanosterol
*
0.49
0.91 f 0.88
25-dihydrolanosterol Total sterols
f
0.2&
0.46 f 0.22 6.24
f
0.44
8.89
f
1.0
5.87
f
0.12
7.03 f 0.19
6.29 f 0.35
3.12 f 1.1
all means calculated from at least three experiments standard deviation detected in one experiment out of three
ty acid composition compared to the wild type. Since approximately 85 to 90 yo of C. albicans fatty acids are found in the phospholipid fraction [16], the total fatty acid composition provides a reasonably accurate reflection of the membrane fatty acid composition. Differences between strains D10 and DlOR were more pronounced involving 16:l content and all C18 fatty acids. Similar differences in mycoses 34, 75-83 (1991)
membrane fatty acid composition of strains D10 and
D 1OR have been previously reported [ 1 11. Most significant was the ratio between 18:l and 18:2. All six strains contained 72-78% C18 fatty acids and the percent saturated fatty acids ranged from 17.218.7 with the exception of strain D10 where 14.3% of the fatty acids were saturated. Potassium emux was examined to ascertain the
M. C. BROUCHTONET AL.
80
Table 5. yo Fatty acid composition of strains of Candida albicans ~~~~
"Y 1
44829
FA
ab2
0.48 f 0.03
I4:O
1.2
0.98 f 0.08
15:O
13
16:O
10
16:1
17:1
f
13
0.8
f
3.0
0.90
f
0.41
3.1
11
f
0.23
f
0.5
f1.0
1.4 f 0.14
0.76 0.69
DlOR
abl f
0.06
0.68
f
0.25
ND
+
0.1
13
f
0.04
D10
0.39 f 0.03
0.52 f 0.08
0.51
0.4W
f
0.06
0.2
12
f
1.2
8.8 f 0.4
8.0 f 0.1
13
f
1.3
1.0 f 0.06
0.92 f 0.15
14
f
3.5
1.2 f 0.31
12
f
1.5
8.8
f
0.4
1.3
f
0.35
5.1
f
0.7
f
0.5
f
0.8
f
J.6
18:l
44
+
2.3
38
+ 2.4
40
f
1.6
42
f
1.4
49
f
1.4
27
f
7.3
18:2
28
f
5.2
32
f
3.4
32
f
1.3
32
f
2.5
22
k 1.6
43
f
5.3
+
0.06
18:O
2.8 f 0.2
0.5
0.35
unknown
yo C18 Yo saturated
75.1 17.4
3.8 f 0.6
0.4 f 0.05
0.77
0.49
1.9
0.58 f 0.10
73.1
75.3
77.7
72.8
75.9
17.2
18.7
17.0
14.3
18.2
ND not detected means were calculated from three independent analyses standard deviation values with no SD are for fatty acids detected in only one analysis of that strain
Table 6.
yo K + Leakage of total cellular K+ of Candida albicans strains in the presence' of amphotericin B and Triton X-100
Treatment
44829
Control
20
.01 pg ml-I
AB
*
3b( 19)'
nYl
ab2
abl
DlOR
23f 6(18)
18f 3(18)
19f 8(19)
39f 5(10)
62f 6(9)
22+ 2(7) 24+ 2(4)
24f 5(7)
22f 3(7) 34f 7(5)
61 f 7(5)
60f 5(5)
31 f 8(5)
53 f 12(8) 51f 7(4)
98 & 13(8) 112f 15(3)
.02 pg ml-I AB
29f 5(7) 34 f lO(5)
.05 pg ml-1AB
39+ 8(4)
32 f lO(4)
39 f lO(4)
47 f 12(5)
.50% Triton
32+ 6(5) 38 f 1 1 (4)
6 6 k 7(4) 60f l(3)
27f 5(5) 30 f lO(4)
22f 5(5) 28f 9(4)
.74% Triton
D10
I
measurements taken after 30 minutes of treatment standard deviation number of individual determinations
-
effects of membrane interactive agents on polyeneresistant mutants with normal and altered membrane sterol composition. This assay is extremely sensitive and results in significant leakage differences at very low concentrations of agent. Table 6 shows the potassium leakage from all strains under control conditions and in the presence of varying concentrations of AB and the detergent, Triton X-100. At 0.01 and 0.02 pg ml-' AB, nyl showed no significant (P< 0.05) increase in K + leakage compared to the control while the wild type strain showed increased leakage at all AB levels. Strain ab2 showed slight but significant leakage at 0.01 and 0.02 pg ml-I AB while strain abl showed a similar result at 0.02 pg ml-' AB. Both strains showed wild type leakage levels at higher drug concentrations. Strain DlOR was considerably more sensitive to AB-induced K+ efflux than wild type strain 44829. Strain D10 was
extremely leaky under control conditions. Leakage was not increased by 0.01 pg ml-I AB while the wildtype revertant strain, DlOR, showed a 50% increase in efnux at the same drug concentration. In the presence of Triton X-100, strain nyl was extremely leaky to K + while a b l and ab2 were less susceptible than the wild type strain. Strain DlOR was moderately affected by the detergent while strain D10 was completely lysed by both concentrations employed. Since sterol mutants of S. cerevisiae have been shown to have altered growth characteristics on a variety of media [ 171, it was of interest to determine whether the membrane alterations represented in the mutants considered in this study also showed such responses. Table 7 shows the mgts of all strains grown with two energy sources, glucose and ethanol, and in the presence and absence of the non-ionic mycoses 34, 75-83 ( 199 1 )
POLYENE RESISTANCE IN ~ A N D I D ALBICANS A
Table 7.
Mean generation times (min) of Candida albicans strains in various media
Media
YPD
44829 96k
nYl 4.7a(6)b
99f
ab2 5.0(5)
76 f
3.9(4)
105k
7.4(4)
69f
D10 6.2(5)
102k
5.7(5) 7.8(5)
YPE
185k 15(5)
176k lO(5)
177f
3.4(4)
163* 15(4)
121?
1.4(3)
129k
146k 15(5)
132k
6(4)
137k
8.6(5)
171k
9.3(4)
102k
5.9(6)
124+ 12(4)
101k
5.4(4)
93k
2.6(3)
87k
7.9(3)
74k
6.6(4)
YPDT YPDET
94k
9.6(4)
152k 16(4)
127k 16(4)
108k 13(4)
168k 18(4)
101k 12(4)
126f
7.1(4)
105+
7.3(3)
standard deviation number of individual determinations
detergent, Tergitol. All strains showed a decreased growth rate when ethanol was substituted for glucose. When both energy sources were present together (YPDE) the growth rates were still reduced indicating that the ethanol effect is due to a combination of its reduced ability to be used as an energy source and its membrane perturbation properties [ 171. D 10 seemed less effected by ethanol than the other strains under all conditions tested. Strain ny 1 showed growth characteristics similar to those of the wild type. Strains abl and ab2 showed growth patterns that were clearly different from that of the wild type strain and different from each other. On glucose, abl is similar to wild type while ab2 grows significantly faster. The addition of Tergitol to YPD had no effect on the growth of 44829, nyl, and DlOR but enhanced the growth of abl while slowing the growth of ab2 and D10. In YPDE medium, Tergitol had no effect on 44829, ny 1, ab 1, and D 1OR but provided a partial protection from ethanol perturbation for ab2 and restored the growth rate ofD10 to the level seen in YPD.
Discussion
'
DlOR
abl
YPDE
a
81
Ergosterol and its biosynthesis represent the two major targets in antifungal drug therapy. The polyene antibiotics specifically bind to membrane ergosterol and disrupt normal membrane function. Much evidence has been accumulated regarding the specific modes of action of these drugs and it appears likely that different polyenes, while interacting with membrane ergosterol, may induce cell death by different mechanisms [18]. Mutations in the ergosterol biosynthetic pathway result in cell membranes where ergosterol is replaced by sterol intermediates. Several such mutants have been isolated in S. cerevisiae based on their resistance to nystatin [9, 101. In comparison, only two types of ergosterol mutants have been conclusively documented in C. albicans [6, 191. One of these, the A8-A7 isomerase is also found in S. cerevisiae [lo] while the second, the mycoses 34, 75-83 (1991)
cytochrome P450-dependent lanosterol demethylase, has not been isolated as a single lesion in this organism. There have been some reports of polyene resistance arising in clinical and laboratory settings that are not the result of lesions in the ergosterol pathway [ l , 71. In these reports and other earlier studies of AB resistance [5, 61, little characterization of the resistant strains was performed beyond MIC determination and a non-quantitative sterol analysis. In this report, two ergosterol-producing, AB-resistant mutants of C. albicans are characterized along with a A8-A7 isomerase mutant and a previously described lanosterol demthylation mutant. In Tables 1 and 2,. the ergosterol-deficient, nystatin-resistant mutants, nyl and D10, are shown to be cross resistant to AB on solid and in liquid media. In contrast, the ergosterol producing, AB-resistant mutants, abl and ab2, show no cross resistance to nystatin in a liquid medium and minimal resistance on a solid medium. This observation further supports the hypothesis that the mechanisms of action of these two polyenes differ [18]. In addition, the susceptibility of abl and ab2 to nystatin argues against a cell wall exclusion mechanism of resistance since it would be unlikely that AB would be excluded while the structurally similar nystatin would not. The sterol compositions exhibited by the mutant strains indicate a wild type profile for abl and ab2 while nyl accumulates a mixture of A* sterols indicating a mutation in the A8-A7 isomerase. This mutant (erg2) has been previously described in S. cerevisiae [ 101 and C. albicans [ 1 1, 191. Strain D10 accumulates exclusively 14-methyl sterols as has been previously reported [12]. There is no qualitative change in sterol content in exponential and stationary phase cultures but total sterol content was greater in stationary phase for all strains as has been described in other yeast strains [20, 211. The wild type sterol composition of strains abl and ab2 is similar to the sterol composition described for strain C7 of C. albicans [ 1 ll. Strain C7
82
M.C.BROUGHTON ET AL.
was selected on the basis of resistance to nystatin. Cross resistance to the polyenes AB and candicidin but not leucomycin was demonstrated for C7 [14]. Although similar in AB resistance and sterol composition to strain C7, abl and ab2 differ from C7 in that they are sensitive to nystatin. Since the AB resistance of abl and ab2 could not be accounted for on the basis of sterol composition, fatty acid composition was examined. Some nystatin-resistant mutants of C. albicans have been shown to have altered fatty acid compositions [l l ]. In addition, non-sterol membrane lipid has been implicated in the interaction of polyenes with membranes [ 1 11. The data presented in Table 5 indicates that, among the major fatty acids, a b l and ab2 show no significant differences compared to the wild type. The % C18 and yo saturated fatty acids were virtually the same also. Fatty acid analysis of strain C7 also showed a composition nearly identical to its wild type [ 1 11. Because analyses of total cellular fatty acids indicated that individual fatty acid composition, yo C18 fatty acids, and yo saturated fatty acids did not differ in abl and ab2 versus the wild type, it is highly unlikely that the phospholipid fatty acid fraction, which comprises 85-90% of total cellular fatty acids, would demonstrate significant fatty acid changes. Of the sterol-based mutants, only D10 demonstrated a fatty acid composition that differed from its wild type. Strain nyl has a wild type fatty acid profile while strain D10 varies significantly from its wild type primarily in the relative percentages of the C18 fatty acids. Since the ergosterol biosynthetic block in this strain occurs so early in the pathway, the cell may compensate for the presence of methylsterols by altering fatty acid composition. Since the analysis of the sterol and fatty acid compositions of strains abl and ab2 showed no significant differences from those of the wild type strain, a more functional property of membranes, that has been shown to be affected by polyene antifungals, was examined. The potassium efflux data shown in Table 6 indicate that at low concentrations of AB (0.01 and 0.02 pg ml-l), abl and ab2 show less leakage than the wild type. This difference is not noted at higher concentrations of AB which is consistent with the low level (relative to nyl and D10) resistance properties presented in Tables 1 and 2. Strain ny 1 shows greater resistance to AB-induced leakage which is also consistent with its level of cross resistance to AB. I n contrast, strains abl and ab2 are more resistant to the membrane disruptive properties of Triton X-100 than the wild type while nyl and D10 are considerably more susceptible to the effects of the detergent. The mechanisms conferring polyene resistance to nyl and the two AB-resistant mutants are also quite different based on membrane susceptibility to perturbators other than polyenes.
The mgt data shown in Table 7 clearly indicate that the membrane alteration resulting from AB resistance effects the physiological capability of cells to grow efficiently under a variety of conditions. nyl, which has a lesion late in the ergosterol pathway, responded similarly to the wild type under the conditions tested. This has been previously reported for the erg2 mutant of S. cerevisiae [17]. D10, an earlier pathway mutant, showed less sensitivity than the wild type to ethanol but appeared more sensitive to Tergitol in a glucose medium. However, the concurrent presence of ethanol and Tergitol (YPDET) produced a normal growth rate. The non-sterol mutants, abl and ab2, are quite different from one another and from the wild type. Compared to the wild type, ab2 had a much faster growth rate on YPD, a reduced growth rate in the presence of Tergitol (YPDT), and showed a protective effect for Tergitol in the presence of ethanol (YPDET). A similar protective effect for Tergitol against the growth reduction properties of ethanol has been described for a C-24 transmethylase mutant of S. cerevisiae [17]. Mutant abl showed a faster growth rate in the presence of Tergitol (YPDT) but Tergitol did not protect this strain against the deleterious effects of ethanol. The potassium leakage data reflecting membrane interaction with AB and Triton X-100 and the growth rate data indicate that the membranes of abl and ab2 differ from those of the wild type. Sterol and fatty acid analyses, however, indicate no significant differences in composition. The resistance mechanism in these two strains may involve subtle changes in the structural features of the membranes which make the membrane ergosterol less accessible to exogenous polyenes. These minor changes in membrane structure may also change the susceptibility of the membrane to other, unrelated membrane active agents. Preliminary studies of membrane fluidity using electron paramagnetic resonance analysis (Lees, unpublished) have indicated that the membranes of ny 1, a b 1, and ab2 have more rigid membranes at one fatty acid carbon position (number 5) than the wild type. This structural alteration has been reported for strain D10 [22] and several of the ergosterol biosynthetic mutants of S. cerevisiae [23] including erg2. Alterations in membrane fluidity have been reported to be linked to changes in many membrane properties including permeability, susceptibility to membrane active agents, and cell growth characteristics [ 17, 24, 251. Although the membrane alterations in ab l and ab2 are not reflected in sterol and fatty acid contents, their effects are noted functionally and structurally. Complete characterization of these functional consequences associated with this type of resistance is warranted in order to devise new ways to deal with non-sterol-related polyene resistance. mycoses 34, 75-83 (1991)
POLYENE RESISTANCE IN ~ A N D I D ALBICANS A
References 1 Bratjburg, J., Powderly, W. G., Kobayashi, G. S. & Medoff,
G. (1990) Amphotericin B: current understanding of mechanisms of action. Antimicrob. Agents Chemother. 34, 183188. 2 Madden, T. D., Janoff, A. S. & Cullis, P. R. (1990) Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatid ylcholine and phosphatidylglycerol. Chem. Phys. Lipids 52, 189-198. 3 Ringel, S. M. (1990) New antifungal agents for the systemic mycoses. Mycopathologia 109, 75-87. 4 Powderly, W. G., Kobayashi, G. S., Herzig, G. P. & Medoff, G. (1988) Amphotericin B-resistant yeast infection in severely immunocompromised patients. Am. J. Med. 84, 826832. 5 Dick, J. D., Merz, W. G. & Saral, R. (1980) Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob. Agents Chemother. 18, 158-163. 6 Hamilton-Miller, J. M. T. (1972) Sterols from polyene-resistant mutants of Candida albicans.J. Gen. Microbiol. 73, 20 1-203. 7 Gale, E. F. (1986) Nature and development of phenotypic resistance to amphotericin B in Candida albicans. Adv. Microb. Physiol. 27, 278-320. 8 Athar, M. A. & Winner, H. L. (1971) The development of resistance by Candida albicans to polyene antibiotics in vitro. J. Med. Microbiol. 4, 505-5 17. 9 Bard, M. (1972) Biochemical and genetic aspects of nystatin resistance in Saccharomycescerevisiae.J. Bacteriol. 111,649657. 10 Molzhan, S. W. & Woods, R. A. (1972) Polyene resistance and the isolation of sterol mutants of Saccharomyces cerevisiae.J. Gen. Microbiol. 72, 339-348. 11 Pierce, A. M., Pierce Jr., H. D., Unrau, A. M. & Oehlschlager, A. C. (1978) Lipid composition and polyene resistance of Candida albicans mutants. Can.J.Biochem. 56, 135142. 12 Bard, M., Lees, N. D., Barbuch, R. J. & Sanglard, D. (1987) Characterization of a cytochrome P450 deficient mutant of Candida albicans. Biochem. Biophys. Res. Commun. 147, 794-800. 13 Kakar, S. N. & Magee, P. T. (1982) Genetic analysis of Candida albicans:identification of different isoleucine-valine, methionine, and arginine alleles by complementation. J. Bacren'ol. 151, 1247-1 252.
rnycoses 34, 75-83 ( 1991 )
83
14 Miller, L. & Berger, T. ( 1 985) Bacterial identification by gas chromatography of whole cell fatty acids. Hewlett Packard Application Note 228-41. Palo Alto, CA: Hewlett Packard co. 15 Bard, M., Albrecht, M. R., Gupta, N., Guynn, C. J. & Stillwell, W. (1988) Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23, 534538. 16 Georgopapadakou, N. H., Dix, B. A., Smith, S. A., Freudenberger, J. & Funke, P. T. (1987) Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candica albicans. Antimicrob. Agents Chemother. 31, 46-51. 17 Lees, N. D., Lofton, S. L., Woods, R. A. & Bard, M. (1980) Effects of vaned energy source and detergent on the growth of sterol mutants of Saccharomyces cerevisiae. J. Gen. Microbiol. 118, 209-214. 18 Thomas, A. H. (1986) Suggested mechanisms for the antimycotic activity of the polyene antibiotics and the N-substituted imidazoles.J. Antimicrob. Chemother. 17, 269-279. 19 Subden, R. E., Safe, L., Moms, D. C., Brown, R. G. & Safe, S. (1977) Eburicol, lichesterol, ergosterol, and obtusifoliol from polyene antibiotic-resistant mutants of Candida albicans. Can.J. Microbiol. 23, 751-754. 20 Parks, L. W. (1978) Metabolism of sterols in yeast. CRC Crit. Rev. Microbiol. 6, 301-341. 21 Woods, R. A., Bard, M., Gardner, I. E. & Molzhan, S. W. (1974) Studies on the accumulation of ergosterol and 24(28)dehydroergosterol in 3 strains of Saccharomyces cerevisiae. Microbios 1 0 4 73-80. 22 Lees, N. D., Kleinhans, F. W., Broughton, M. C., Pennington, D. E., Ricker, V. A. & Bard, M. (1989) Membrane fluidity alterations in a cytochrome P450-deficient mutant of Candida albicans. Steroids 53, 567-578. 23 Lees, N. D., Bard, M., Kemple, M. D., Haak, R. A. & Kleinhans, F. W. (1979) ESR determination of membrane order parameter in yeast sterol mutants. Biochim. Biophys. Acta 553, 469-475. 24 Bard, M., Lees, N. D., Burrows, L. S., Kleinhans, F. W. (1978) Differencesin crystal violet uptake and cation-induced death among yeast sterol mutants. J. Bacteriol. 135, 11461148. 25 Lees, N. D., Broughton, M. C., Sanglard, D. & Bard, M. (1990) h o l e susceptibility and hyphal formation in a cytochrome P-450-deficient mutant of Candida albicans. Antimicrob. Agents Chemother. -83 1-836.
ACCEPTED: JANUARY 8, 1991
34, 75-83 (1991)
Polyene resistance in ergosterol producing strains of Candida albicans Polyen-Resistenz bei Ergosterin-produzierenden Candida albicansStammen M. Christine Broughton, M. Bard and N. D. Lees Key words. Candida albicans, polyene resistance, amphotericin B resistance. Schliisselworter. Candida albicans, pol yen-Resistenz, Amphotericin B-Resistenz.
Summary. Following nitrous acid mutagenesis, one nystatin- (nyl) and two amphotericin B (AB)resistant (abl and ab2) mutants of Candida albicans were isolated and characterized. The three mutants plus a previously described cytochrome P450deficient mutant (D10) of this organism were analyzed for polyene cross resistance. Cross resistance was noted for nyl and D10 but not for abl and ab2. Sterol analysis indicated that nyl was a A8-A7 isomerase mutant while abl and ab2 showed wild type sterol profiles. Fatty acid analysis showed no significant differences for abl, ab2, and nyl compared to wild type while D 10 showed more pronounced differences. AB- and Triton X-1 00-induced potassium leakage studies indicated that abl and ab2 are resistant to low AB levels and nyl is resistant to higher AB levels. In contrast, a b l and ab2 were more resistant to detergent-induced potassium leakage than the wild type or mutants nyl and DlO. Significant differences in growth rate, ethanol sensitivity, and response to Tergitol were also noted among the resistant strains. The data indicate a different mechanism of action for the two polyenes and indicate a resistance mechanism for abl and ab2 based on subtle alterations of membrane structure rather than sterol substitution.
Department of Biology, Indiana University - Purdue University at Indianapolis, Indiana, USA. Correspondence: Professor Dr Norman D. Lees, Department of Biology, Indiana University- Purdue University, 1125 East 38th Street, Indianapolis, IN 46205, USA.
Zusammenfassung. Im AnschluR an eine Mu-
tagenese mit salpetriger Saure wurden Mutanten von Candida albicans isoliert und charakterisiert, eine Nystatin- (nyl) resistente und zwei Amphotericin B (AB)-resistente (abl und ab2). Die drei Mutanten und eine andere, Zytochrom P450-defizitare Mutante (D10) dieses Organismus, welche fruher beschrieben wurde, wurden auf Polyen-Kreuzresistenz analysiert. Kreuzresistenz wurde fur ny 1 und D10 festgestellt, aber nicht fur abl und ab2. Eine Sterinanalyse ergab, daI3 ny 1 eine A8-A7-IsomeraseMutante war, wahrend abl und ab2 Wildtyp-Sterinprofile aufwiesen. Eine Fettsaurenanalyse zeigte keine signifikanten Unterschiede fur abl, ab2 und nyl im Vergleich zu Wildtypen. D10 hingegen wies ausgepragtere Unterschiede auf. Untersuchungen der Kaliumdurchlassigkeit, induziert durch ABund Triton X-100, ergaben, daR abl und ab2 gegen ein niedriges, nyl aber gegen ein hoheres AB-Niveau resistent sind. Im Gegensatz d a m hatten abl und ab2 eine hohere Resistenz gegen eine durch Detergenzien induzierte Kaliumdurchlassigkeit als die Wildtypen oder Mutanten nyl und D10. Signifikante Unterschiede in der Wachstumsrate, Athylalkohol-Sensitivitat und Reaktion auf Tergitol wurden unter den resistenten Stammen ebenfalls festgestellt. Die Daten weisen auf einen unterschiedlichen Wirkungsmechanismus fur die beiden Polyene hin sowie auf einen Resistenzmechanismus fur abl und ab2, welcher eher auf einer subtilen Anderung der Membranstruktur als auf einer SterinSubsti tution beruht.
76
M.C . BROUGHTON ET AL.
Introduction
Methods
Amphotericin B (AB) is an effective antifungal agent that has an extended history of successful use in systemic fungal infections. There are, however, a number of serious side effects [ 13 associated with this drug which have limited its usefulness and led to the development of alternative ways in which to administer the drug [2] and to the development of new antifungal agents [3]. Although resistance to AB has not been frequently reported for clinical isolates, recently the appearance of low level AB resistance in a variety of pathogenic yeasts including C. albicans has been documented in severely immunocompromised patients [4]. In this study [4], as in past studies [5, 61, a thorough analysis of the resistance mechanism was not undertaken. These limited clinical studies, plus more extensive studies of laboratory isolates of ABresistant strains, have resulted in several possible mechanism of AB resistance including cell wall exclusion [7], increased ergosterol content [6], and reduced ergosterol content [8-101. The latter mechanism is most consistent with the extensive data collected using AB, and other polyenes including nystatin, which indicate a relationship between drug affinity for ergosterol, the major fungal membrane sterol, and antifungal activity [ 13. This relationship has been employed in experiments in which ergosterol biosynthetic mutants have been isolated in fungi, including Candida albicans [l 11 and Saccharomyces cerevisiae [9, 101, based on resistance to polyenes. There have been a few reports of low level AB resistance, from clinical and laboratory studies, in which the resistance mechanism seems to be related to factors other than ergosterol content [ 1, 71. Most of these studies go only as far as determining the minimal inhibitory concentrations of AB and performing a simple analysis of cellular sterol content. Resistance to low levels of AB is of great clinical significance since resistance levels as low as 0.8 pg ml-I have been shown to result in patient fatality [4]. The mechanism associated with and the corresponding physiological characteristics of such mutants should be of importance in drug strategies to combat future resistance which seems likely based on the increase in infections with C. albicans [4]. In this study we will describe the characteristics of several polyene resistant mutants of C. albicans, some of which have wild type sterol profiles.
Fungi Nystatin- and AB-resistant mutants were derived from C. albicans strain ATCC 44829. C. albicans strain D10 (ATCC 38247) a nystatin-resistant, cytochrome P450-deficient mutant, has been previously described [ 11, 121. Strain DlOR is a wild type revertant of strain D10 isolated as a fast growing colony in a lawn of D10 on a complete medium without nystatin [12].
Media and growth conditions Cells were grown in 1% Difco yeast extract, 2% Difco peptone, and 2% glucose (YPD). 2% ethanol was used to replace glucose (YPE) or added in addition to glucose (YPDE). The non-ionic detergent, Tergitol NP-40 (Sigma), was added at a concentration of 1.5%. Plates were made by adding 2% agar. Strain D10 was maintained on YPD plates supplemented with 35 pg ml-' nystatin. AB-resistant mutants a b l and ab2 were maintained on YPD plates supplemented with 3 and 1 pg ml-' of AB, respectively. All growth was performed at 30 "C.
An tifungal agen ts Nystatin and AB were obtained from Sigma. Nystatin stocks were prepared at 4 mg ml-I in dimethylformamide and stored a t - 20 "C. AB stocks were prepared at 5 mg ml-I in dimethylformamide and stored at - 20 "C.
Mutagenesis Nitrous acid mutagenesis was performed according to the procedure of Kakar & Magee [ 131. Samples of YPD cultures were collected by centrifugation, washed and resuspended in 0.1 mol sodium acetate buffer, pH 4.5, containing 30 mmol (abl and ab2) or 60 mmol (nyl) sodium nitrite. After 5 min (abl and ab2) or 60 min (nyl) of incubation, the cells were collected by centrifugation and resuspended in 0.1 mol phosphate buffer, pH 7.5, to stop the mutagenesis. The suspensions were transferred to sterile, plastic petri dishes and exposed to short wave ultraviolet irradiation for 15 sec (abl and ab2) or 45 sec (nyl) to promote chromosomal crossing over. For the isolation of AB-resistant mutants, rnutagenized cells were grown overnight in YPD supplemented with 6 pg ml-' AB. Cells from the overnight growth were then plated on YPD supplemented with the same concentration of AB. For the isolation of nystatin-resistant mutants, mutagenized cells were plated directly on YPD supplemented with 5 pg ml-' nystatin. mycoses 34, 75-83 ( 1991)
POLYENERESISTANCE
Minim urn Inhibitory Concentration (MIC) For the determination of MIC on a solid medium, overnight YPD broth cultures were diluted in 0.9% saline to lo3 cells/ml. 0.1 ml of the dilution was spread on a YPD plate supplemented with AB or nystatin. MIC was defined as the lowest concentration at which no growth occurred after 48 h of incubation. For the determination of MIC in a liquid medium, cells from overnight YPD broth cultures were used to inoculate 40 ml of YPD broth supplemented with AB or nystatin. Initial turbidities were 8-15 Klett units as determined using a Klett Summerson colorimeter with a no. 66 red filter. MIC was defined as the lowest concentration at which no increase in turbidity was noted after 24 h of incubation.
Sterol analysis Quantitative sterol analysis was determined for exponential phase cells by inoculating YPD broth with cells from an overnight YPD plate and growing to early exponential phase (20-60 Klett units). 200 ml samples were removed for sterol analysis and 40 ml samples were transferred to tared centrifuge tubes for dry weight determinations. For stationary phase cultures, YPD broth cultures were grown for 48 h. 25 ml samples were removed for sterol analysis and 10 ml samples were transferred to tared centrifuge tubes for dry weight determinations. Dry weights were determined by drying the cell pellets for 48 h at 62 "C and reweighing the tared tubes. Samples for sterol analysis were collected by centrifugation and cells resuspended in 40% alcoholic KOH (25 g K O H dissolved in 36 ml water, brought to 100 ml total volume with 95% ethanol) for saponification. The suspension was transferred to a round bottom flask, a boiling chip added and a reflux tube attached, before being placed in an 8590°C water bath for 2 h. After 1 h, 1 ml of 95% ethanol was added to replace volume lost by evaporation. The flask was removed from the bath and allowed to cool. Water (4 ml) and heptane (10 ml) were added and the mixture vigorously shaken for 3 min. The flask was stored at room temperature for 15 min to allow separation of the organic and aqueous phases. The organic phase was removed for sterol analysis. Sterol analysis was performed on an H P 5710A gas chromatograph using an SE-30 six foot glass column at 240 "C. The carrier gas was nitrogen at 60 ml/min. Five pl of the heptane phase was used for each analysis. Peak retention times and areas were determined with an H P 3392A integrator. Sterol concentrations were determined using ergosterol (0.1 mg ml-' heptane; Sigma) and lanosterol rnycoses 34, 75-83 (199 1)
IN
CANDIDA ALBICANS
77
(0.1 mg ml-' heptane; Sigma) standards' peak areas as references.
Fatty acid analysis Fatty acid analysis was performed according to the procedure of Miller & Berger [ 141. A loopful of cells from an overnight YPD plate was used to coat the bottom of a screw cap glass tube. One ml of saponification reagent (45 g NaOH, 50 ml methanol, and 150 ml water) was added and the tube vortexed. The tube was incubated at 100°C for 5 min, vortexed, and reincubated at 100 "C for 25 min. After cooling in a water bath, 2 ml of methylating reagent (325 m l 6 N HCl and 275 ml methanol) was added and the mixture vortexed prior to incubation at 80 "C for 10 min. 1.25 ml of extraction reagent (1:l ratio of hexane and methyl-t-butyl-ether) was added and the contents of the tube mixed by inversion. Upon phase separation, the bottom aqueous phase was removed. 3 ml of wash reagent (10.8 g NaOH in 900 ml water) was added to the remaining organic phase to remove the fatty acids and the tube was inverted several times for 5 min followed by the addition of 2-3 drops of saturated NaCl to aid phase separation. Approximately 2/3 of the upper, organic phase was removed for fatty acid analysis. Fatty acid analysis was performed using 2 pl samples on an H P 5890GC gas chromatograph using a 25 m x 0.2 mm methyl phenyl silicone fused silica capillary column at 250°C. The carrier gas was nitrogen at 30 ml/min. Fatty acids were identified by relative retention times compared to fatty acid standards and quantitated as yo total fatty acids.
Potassium leakage YPD broth was inoculated from an overnight YPD plate to a concentration of 8-15 Klett units and grown to exponential phase. Cells were prepared, treated, and assayed for potassium leakage as dscribed by Bard et al. [ 151. Cells were collected by centrifugation and washed twice and resuspended in 10 mmol Tris, 0.9% NaCl buffer, pH 7.0, to a final concentration of 3 x lo7 cells/ml. Samples (treated and untreated) were incubated at 30 "C on a shaking water bath a t 200 rpm for 30 min. Cells were removed by centrifugation and the supernatant fluids were assayed on an IL 952 atomic absorption spectrophotometer to determine potassium concentration. Total cellular potassium was determined by boiling a sample for 10-15 min to lyse the cells. One tailed t-tests were performed to determine the statistical significance between critical data points. Probabilities greater than 0.05 were not considered significant.
78
M. C . BROUGHTON
ET AL.
Mean generation time (mgt)
Table 2.
Cells from overnight cultures grown in YPD broth were collected by centrifugation and resuspended at a concentration of 8-15 Klett units in a 250 ml nephelometer flask containing 20 ml of the medium to be tested. Cells were incubated at 30 "C and turbidity monitored at appropriate intervals. The mgt values were calculated during exponential growth phase.
Results Nitrous acids mutagenesis of C. albicans strain 44829 followed by UV irradiation resulted in the isolation of one nystatin-resistant mutant (nyl) and four AB-resistant mutants (abl, ab2, ab3, and ab4). Initial characterization of resistance levels indicated that abl was more resistant (8 times that of wild type) than the other three isolates (3 times that of wild type). Thus, abl and one of the lesser resistant strains, ab2, were chosen for further characterization. In addition, strain D10 of C. albicans, a nystatin-resistant, cytochrome P450-deficient mutant [ l l , 121 and its wild type revertant, strain DlOR, were included in the characterization since this particular mutation represents one of only two verified steps in ergosterol biosynthesis for which there are blocks in this organism. The results of MIC determinations on a solid medium are shown in Table 1. The level of nystatin resistance of strain ny 1 was significantly higher than that seen in the wild type but far below that seen for strain D10. Both of these mutants showed cross resistance to AB. The AB-resistant strains, a b l and ab2, showed 8 and 3 times, respectively, the level of AB resistance seen in the wild type. Both strains showed minimal cross resistance to nystatin. The MIC data in a liquid medium shown in Table 2 indicate a similar but more pronounced pattern of AB resistance. Nystatin resistance levels for Table 1. Minimum inhibitory concentrations*of polyene antibiotics for Candida albicans strains grown on solid media for 48 hours Antibiotic
44829 nyl
Nystatin 3.5b (Pip ml-9 Amphotericin B 2.0 (pg ml-')
ab2
abl
D10
DlOR
25.0
5.0
5.0
75.0
3.0
5.0
6.0
16.0
30.0
1.5
' lowest concentration of antibiotic necessary to completely inhibit growth data represented as means of 2 or 3 experiments. Variation among experiments was 1 pg ml-' or less for all entries
Minimum inhibitory concentrationsaof polyene antibiotics for Candida albicans strains grown in liquid media for 24 hours
I Antibiotic
~~
DlOR
44829 nyl
ab2
abl
D10
10.0
40.0
10.0
10.0
150.0 5.0
Amphotericin B 1.0 ( M ml-9
25.0
3.0
5.0
150.0 1.0
I
Nystatin
(Md-9 I
a
lowest concentration of antibiotic necessary to completely inhibit growth data represented as means of 2 or 3 experiments. Variation among experiments was 1 pg ml-1 or less for all entries
all strains were uniformly higher. Strain D10 showed very high resistance while the level for strain nyl was four times that of its wild type. Both of these strains showed significantly greater cross resistance to AB compared to the solid medium MIC results. With the exceptions of strains D10 and nyl, AB resistance levels were lower in the liquid medium compared to the solid medium. In addition, ab l and ab2 showed no cross resistance to nystatin. Sterol analysis of exponential phase cells, shown in Table 3, indicated a typical, wild type sterol profile for strains 44829 and DlOR, with ergosterol present as the dominant sterol and low levels of lanosterol [5, 11, 121. Previously, more extensive analysis (gas chromatography-mass spectral analysis) of strain DlOR [12] indicated the presence of two additional minor sterols that were identified as ergosta-dien-3P-01s. Strain nyl showed no detectable egosterol. Instead, it accumulated a mixture of delta-8 sterols, that have since been identified (Barbuch et al., unpublished) as ergosta-5,8,22-trien-3P01, ergosta-8-en-3P-01, and fecosterol. This sterol composition is consistent with that reported for the erg2 mutant of S. cerevisiae [9,101 and the E4 mutant of C. albicans [l l ]. Strain D10 accumulated the five methyl sterols previously reported [ 121. Strains abl and ab2 produced sterol profiles essentially identical to that of the wild type strain. Total sterol content was 3 0 4 0 % higher for nyl compared to the wild types and the AB-resistant strains while strain D10 produced considerably less sterol. In general, sterol analysis of stationary phase cells (Table 4) indicated uniformly higher sterol content and profiles similar to those noted in exponential phase cells for all strains. Exceptions, were a significant increase in ergosta-5,8,22-trien-3P-ol for ny 1 and an elevated ergosterol level in abl. Since some nystatin-resistant mutants of S. cerevisiae showed altered fatty acid composition [ 1 13, this feature of the strains was examined. The data in Table 5 indicate that strains nyl, ab2, and abl showed no significant difference in total cellular fatmycoses 34, 75-83 (1991)
POLYENE RESISTANCE IN GNDIDA ALBICANS
I Table 3.
79
Sterol content (pg sterol/mg dry weight) of exponential phase cultures of Candida albicans strains Strains
Sterol
44829
nYl
ab2
abl
DlOR
4.12f0.12
4.03f0.66
3.97k0.72
D10
I
1.45ak0.13b
ergosta-5,8, 22-triene-3P-01 3.81 k 0.38
ergosterol
0.60f0.16
14-methylfecosterol 4.32 a 0.27
ergosta-8-en-3P-01 & fecosterol
0.42 f 0.13
obtusifoliol 0.16 f 0.02
lanosterol
0.27k0.09
0.34k0.05
0.26k0.02
24-meth ylene-24, 25-dihydrolanosterol
0.42 f 0.17 0.03 f 0.02
25-dih ydrolanosterol Total sterols a
0.37f0.15
3.97f0.32
5.77f0.38
4.29f0.20
4.27k0.70
4.23k0.74
1.84k0.52
all means calculated from at least three experiments standard deviation
I Table 4.
Sterol content (pg sterol/mg dry weight) of stationary phase cultures of Candida albicans strains Strains
Sterol
"Y 1
44829
ab2
abl
DlOR
D10
I
ergosta-5,8, 22-triene-3P-ol ergosterol
4.12' k 0.45b 5.84 f 0.38
5.41
f
0.17
6.29
f
0.19
6.21
f
0.33 0.46 f 0.31
14-methylfecosterol 4.77
ergosta-8-en-3P-01 & fecosterol
f
0.69
obtusifoliol lanosterol
1.01 0.40 f 0.07
0.46 f 0.07
0.74 f 0.01
0.08 f 0.06
24-methy he-24, 25-dihydrolanosterol
*
0.49
0.91 f 0.88
25-dihydrolanosterol Total sterols
f
0.2&
0.46 f 0.22 6.24
f
0.44
8.89
f
1.0
5.87
f
0.12
7.03 f 0.19
6.29 f 0.35
3.12 f 1.1
all means calculated from at least three experiments standard deviation detected in one experiment out of three
ty acid composition compared to the wild type. Since approximately 85 to 90 yo of C. albicans fatty acids are found in the phospholipid fraction [16], the total fatty acid composition provides a reasonably accurate reflection of the membrane fatty acid composition. Differences between strains D10 and DlOR were more pronounced involving 16:l content and all C18 fatty acids. Similar differences in mycoses 34, 75-83 (1991)
membrane fatty acid composition of strains D10 and
D 1OR have been previously reported [ 1 11. Most significant was the ratio between 18:l and 18:2. All six strains contained 72-78% C18 fatty acids and the percent saturated fatty acids ranged from 17.218.7 with the exception of strain D10 where 14.3% of the fatty acids were saturated. Potassium emux was examined to ascertain the
M. C. BROUCHTONET AL.
80
Table 5. yo Fatty acid composition of strains of Candida albicans ~~~~
"Y 1
44829
FA
ab2
0.48 f 0.03
I4:O
1.2
0.98 f 0.08
15:O
13
16:O
10
16:1
17:1
f
13
0.8
f
3.0
0.90
f
0.41
3.1
11
f
0.23
f
0.5
f1.0
1.4 f 0.14
0.76 0.69
DlOR
abl f
0.06
0.68
f
0.25
ND
+
0.1
13
f
0.04
D10
0.39 f 0.03
0.52 f 0.08
0.51
0.4W
f
0.06
0.2
12
f
1.2
8.8 f 0.4
8.0 f 0.1
13
f
1.3
1.0 f 0.06
0.92 f 0.15
14
f
3.5
1.2 f 0.31
12
f
1.5
8.8
f
0.4
1.3
f
0.35
5.1
f
0.7
f
0.5
f
0.8
f
J.6
18:l
44
+
2.3
38
+ 2.4
40
f
1.6
42
f
1.4
49
f
1.4
27
f
7.3
18:2
28
f
5.2
32
f
3.4
32
f
1.3
32
f
2.5
22
k 1.6
43
f
5.3
+
0.06
18:O
2.8 f 0.2
0.5
0.35
unknown
yo C18 Yo saturated
75.1 17.4
3.8 f 0.6
0.4 f 0.05
0.77
0.49
1.9
0.58 f 0.10
73.1
75.3
77.7
72.8
75.9
17.2
18.7
17.0
14.3
18.2
ND not detected means were calculated from three independent analyses standard deviation values with no SD are for fatty acids detected in only one analysis of that strain
Table 6.
yo K + Leakage of total cellular K+ of Candida albicans strains in the presence' of amphotericin B and Triton X-100
Treatment
44829
Control
20
.01 pg ml-I
AB
*
3b( 19)'
nYl
ab2
abl
DlOR
23f 6(18)
18f 3(18)
19f 8(19)
39f 5(10)
62f 6(9)
22+ 2(7) 24+ 2(4)
24f 5(7)
22f 3(7) 34f 7(5)
61 f 7(5)
60f 5(5)
31 f 8(5)
53 f 12(8) 51f 7(4)
98 & 13(8) 112f 15(3)
.02 pg ml-I AB
29f 5(7) 34 f lO(5)
.05 pg ml-1AB
39+ 8(4)
32 f lO(4)
39 f lO(4)
47 f 12(5)
.50% Triton
32+ 6(5) 38 f 1 1 (4)
6 6 k 7(4) 60f l(3)
27f 5(5) 30 f lO(4)
22f 5(5) 28f 9(4)
.74% Triton
D10
I
measurements taken after 30 minutes of treatment standard deviation number of individual determinations
-
effects of membrane interactive agents on polyeneresistant mutants with normal and altered membrane sterol composition. This assay is extremely sensitive and results in significant leakage differences at very low concentrations of agent. Table 6 shows the potassium leakage from all strains under control conditions and in the presence of varying concentrations of AB and the detergent, Triton X-100. At 0.01 and 0.02 pg ml-' AB, nyl showed no significant (P< 0.05) increase in K + leakage compared to the control while the wild type strain showed increased leakage at all AB levels. Strain ab2 showed slight but significant leakage at 0.01 and 0.02 pg ml-I AB while strain abl showed a similar result at 0.02 pg ml-' AB. Both strains showed wild type leakage levels at higher drug concentrations. Strain DlOR was considerably more sensitive to AB-induced K+ efflux than wild type strain 44829. Strain D10 was
extremely leaky under control conditions. Leakage was not increased by 0.01 pg ml-I AB while the wildtype revertant strain, DlOR, showed a 50% increase in efnux at the same drug concentration. In the presence of Triton X-100, strain nyl was extremely leaky to K + while a b l and ab2 were less susceptible than the wild type strain. Strain DlOR was moderately affected by the detergent while strain D10 was completely lysed by both concentrations employed. Since sterol mutants of S. cerevisiae have been shown to have altered growth characteristics on a variety of media [ 171, it was of interest to determine whether the membrane alterations represented in the mutants considered in this study also showed such responses. Table 7 shows the mgts of all strains grown with two energy sources, glucose and ethanol, and in the presence and absence of the non-ionic mycoses 34, 75-83 ( 199 1 )
POLYENE RESISTANCE IN ~ A N D I D ALBICANS A
Table 7.
Mean generation times (min) of Candida albicans strains in various media
Media
YPD
44829 96k
nYl 4.7a(6)b
99f
ab2 5.0(5)
76 f
3.9(4)
105k
7.4(4)
69f
D10 6.2(5)
102k
5.7(5) 7.8(5)
YPE
185k 15(5)
176k lO(5)
177f
3.4(4)
163* 15(4)
121?
1.4(3)
129k
146k 15(5)
132k
6(4)
137k
8.6(5)
171k
9.3(4)
102k
5.9(6)
124+ 12(4)
101k
5.4(4)
93k
2.6(3)
87k
7.9(3)
74k
6.6(4)
YPDT YPDET
94k
9.6(4)
152k 16(4)
127k 16(4)
108k 13(4)
168k 18(4)
101k 12(4)
126f
7.1(4)
105+
7.3(3)
standard deviation number of individual determinations
detergent, Tergitol. All strains showed a decreased growth rate when ethanol was substituted for glucose. When both energy sources were present together (YPDE) the growth rates were still reduced indicating that the ethanol effect is due to a combination of its reduced ability to be used as an energy source and its membrane perturbation properties [ 171. D 10 seemed less effected by ethanol than the other strains under all conditions tested. Strain ny 1 showed growth characteristics similar to those of the wild type. Strains abl and ab2 showed growth patterns that were clearly different from that of the wild type strain and different from each other. On glucose, abl is similar to wild type while ab2 grows significantly faster. The addition of Tergitol to YPD had no effect on the growth of 44829, nyl, and DlOR but enhanced the growth of abl while slowing the growth of ab2 and D10. In YPDE medium, Tergitol had no effect on 44829, ny 1, ab 1, and D 1OR but provided a partial protection from ethanol perturbation for ab2 and restored the growth rate ofD10 to the level seen in YPD.
Discussion
'
DlOR
abl
YPDE
a
81
Ergosterol and its biosynthesis represent the two major targets in antifungal drug therapy. The polyene antibiotics specifically bind to membrane ergosterol and disrupt normal membrane function. Much evidence has been accumulated regarding the specific modes of action of these drugs and it appears likely that different polyenes, while interacting with membrane ergosterol, may induce cell death by different mechanisms [18]. Mutations in the ergosterol biosynthetic pathway result in cell membranes where ergosterol is replaced by sterol intermediates. Several such mutants have been isolated in S. cerevisiae based on their resistance to nystatin [9, 101. In comparison, only two types of ergosterol mutants have been conclusively documented in C. albicans [6, 191. One of these, the A8-A7 isomerase is also found in S. cerevisiae [lo] while the second, the mycoses 34, 75-83 (1991)
cytochrome P450-dependent lanosterol demethylase, has not been isolated as a single lesion in this organism. There have been some reports of polyene resistance arising in clinical and laboratory settings that are not the result of lesions in the ergosterol pathway [ l , 71. In these reports and other earlier studies of AB resistance [5, 61, little characterization of the resistant strains was performed beyond MIC determination and a non-quantitative sterol analysis. In this report, two ergosterol-producing, AB-resistant mutants of C. albicans are characterized along with a A8-A7 isomerase mutant and a previously described lanosterol demthylation mutant. In Tables 1 and 2,. the ergosterol-deficient, nystatin-resistant mutants, nyl and D10, are shown to be cross resistant to AB on solid and in liquid media. In contrast, the ergosterol producing, AB-resistant mutants, abl and ab2, show no cross resistance to nystatin in a liquid medium and minimal resistance on a solid medium. This observation further supports the hypothesis that the mechanisms of action of these two polyenes differ [18]. In addition, the susceptibility of abl and ab2 to nystatin argues against a cell wall exclusion mechanism of resistance since it would be unlikely that AB would be excluded while the structurally similar nystatin would not. The sterol compositions exhibited by the mutant strains indicate a wild type profile for abl and ab2 while nyl accumulates a mixture of A* sterols indicating a mutation in the A8-A7 isomerase. This mutant (erg2) has been previously described in S. cerevisiae [ 101 and C. albicans [ 1 1, 191. Strain D10 accumulates exclusively 14-methyl sterols as has been previously reported [12]. There is no qualitative change in sterol content in exponential and stationary phase cultures but total sterol content was greater in stationary phase for all strains as has been described in other yeast strains [20, 211. The wild type sterol composition of strains abl and ab2 is similar to the sterol composition described for strain C7 of C. albicans [ 1 ll. Strain C7
82
M.C.BROUGHTON ET AL.
was selected on the basis of resistance to nystatin. Cross resistance to the polyenes AB and candicidin but not leucomycin was demonstrated for C7 [14]. Although similar in AB resistance and sterol composition to strain C7, abl and ab2 differ from C7 in that they are sensitive to nystatin. Since the AB resistance of abl and ab2 could not be accounted for on the basis of sterol composition, fatty acid composition was examined. Some nystatin-resistant mutants of C. albicans have been shown to have altered fatty acid compositions [l l ]. In addition, non-sterol membrane lipid has been implicated in the interaction of polyenes with membranes [ 1 11. The data presented in Table 5 indicates that, among the major fatty acids, a b l and ab2 show no significant differences compared to the wild type. The % C18 and yo saturated fatty acids were virtually the same also. Fatty acid analysis of strain C7 also showed a composition nearly identical to its wild type [ 1 11. Because analyses of total cellular fatty acids indicated that individual fatty acid composition, yo C18 fatty acids, and yo saturated fatty acids did not differ in abl and ab2 versus the wild type, it is highly unlikely that the phospholipid fatty acid fraction, which comprises 85-90% of total cellular fatty acids, would demonstrate significant fatty acid changes. Of the sterol-based mutants, only D10 demonstrated a fatty acid composition that differed from its wild type. Strain nyl has a wild type fatty acid profile while strain D10 varies significantly from its wild type primarily in the relative percentages of the C18 fatty acids. Since the ergosterol biosynthetic block in this strain occurs so early in the pathway, the cell may compensate for the presence of methylsterols by altering fatty acid composition. Since the analysis of the sterol and fatty acid compositions of strains abl and ab2 showed no significant differences from those of the wild type strain, a more functional property of membranes, that has been shown to be affected by polyene antifungals, was examined. The potassium efflux data shown in Table 6 indicate that at low concentrations of AB (0.01 and 0.02 pg ml-l), abl and ab2 show less leakage than the wild type. This difference is not noted at higher concentrations of AB which is consistent with the low level (relative to nyl and D10) resistance properties presented in Tables 1 and 2. Strain ny 1 shows greater resistance to AB-induced leakage which is also consistent with its level of cross resistance to AB. I n contrast, strains abl and ab2 are more resistant to the membrane disruptive properties of Triton X-100 than the wild type while nyl and D10 are considerably more susceptible to the effects of the detergent. The mechanisms conferring polyene resistance to nyl and the two AB-resistant mutants are also quite different based on membrane susceptibility to perturbators other than polyenes.
The mgt data shown in Table 7 clearly indicate that the membrane alteration resulting from AB resistance effects the physiological capability of cells to grow efficiently under a variety of conditions. nyl, which has a lesion late in the ergosterol pathway, responded similarly to the wild type under the conditions tested. This has been previously reported for the erg2 mutant of S. cerevisiae [17]. D10, an earlier pathway mutant, showed less sensitivity than the wild type to ethanol but appeared more sensitive to Tergitol in a glucose medium. However, the concurrent presence of ethanol and Tergitol (YPDET) produced a normal growth rate. The non-sterol mutants, abl and ab2, are quite different from one another and from the wild type. Compared to the wild type, ab2 had a much faster growth rate on YPD, a reduced growth rate in the presence of Tergitol (YPDT), and showed a protective effect for Tergitol in the presence of ethanol (YPDET). A similar protective effect for Tergitol against the growth reduction properties of ethanol has been described for a C-24 transmethylase mutant of S. cerevisiae [17]. Mutant abl showed a faster growth rate in the presence of Tergitol (YPDT) but Tergitol did not protect this strain against the deleterious effects of ethanol. The potassium leakage data reflecting membrane interaction with AB and Triton X-100 and the growth rate data indicate that the membranes of abl and ab2 differ from those of the wild type. Sterol and fatty acid analyses, however, indicate no significant differences in composition. The resistance mechanism in these two strains may involve subtle changes in the structural features of the membranes which make the membrane ergosterol less accessible to exogenous polyenes. These minor changes in membrane structure may also change the susceptibility of the membrane to other, unrelated membrane active agents. Preliminary studies of membrane fluidity using electron paramagnetic resonance analysis (Lees, unpublished) have indicated that the membranes of ny 1, a b 1, and ab2 have more rigid membranes at one fatty acid carbon position (number 5) than the wild type. This structural alteration has been reported for strain D10 [22] and several of the ergosterol biosynthetic mutants of S. cerevisiae [23] including erg2. Alterations in membrane fluidity have been reported to be linked to changes in many membrane properties including permeability, susceptibility to membrane active agents, and cell growth characteristics [ 17, 24, 251. Although the membrane alterations in ab l and ab2 are not reflected in sterol and fatty acid contents, their effects are noted functionally and structurally. Complete characterization of these functional consequences associated with this type of resistance is warranted in order to devise new ways to deal with non-sterol-related polyene resistance. mycoses 34, 75-83 (1991)
POLYENE RESISTANCE IN ~ A N D I D ALBICANS A
References 1 Bratjburg, J., Powderly, W. G., Kobayashi, G. S. & Medoff,
G. (1990) Amphotericin B: current understanding of mechanisms of action. Antimicrob. Agents Chemother. 34, 183188. 2 Madden, T. D., Janoff, A. S. & Cullis, P. R. (1990) Incorporation of amphotericin B into large unilamellar vesicles composed of phosphatid ylcholine and phosphatidylglycerol. Chem. Phys. Lipids 52, 189-198. 3 Ringel, S. M. (1990) New antifungal agents for the systemic mycoses. Mycopathologia 109, 75-87. 4 Powderly, W. G., Kobayashi, G. S., Herzig, G. P. & Medoff, G. (1988) Amphotericin B-resistant yeast infection in severely immunocompromised patients. Am. J. Med. 84, 826832. 5 Dick, J. D., Merz, W. G. & Saral, R. (1980) Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob. Agents Chemother. 18, 158-163. 6 Hamilton-Miller, J. M. T. (1972) Sterols from polyene-resistant mutants of Candida albicans.J. Gen. Microbiol. 73, 20 1-203. 7 Gale, E. F. (1986) Nature and development of phenotypic resistance to amphotericin B in Candida albicans. Adv. Microb. Physiol. 27, 278-320. 8 Athar, M. A. & Winner, H. L. (1971) The development of resistance by Candida albicans to polyene antibiotics in vitro. J. Med. Microbiol. 4, 505-5 17. 9 Bard, M. (1972) Biochemical and genetic aspects of nystatin resistance in Saccharomycescerevisiae.J. Bacteriol. 111,649657. 10 Molzhan, S. W. & Woods, R. A. (1972) Polyene resistance and the isolation of sterol mutants of Saccharomyces cerevisiae.J. Gen. Microbiol. 72, 339-348. 11 Pierce, A. M., Pierce Jr., H. D., Unrau, A. M. & Oehlschlager, A. C. (1978) Lipid composition and polyene resistance of Candida albicans mutants. Can.J.Biochem. 56, 135142. 12 Bard, M., Lees, N. D., Barbuch, R. J. & Sanglard, D. (1987) Characterization of a cytochrome P450 deficient mutant of Candida albicans. Biochem. Biophys. Res. Commun. 147, 794-800. 13 Kakar, S. N. & Magee, P. T. (1982) Genetic analysis of Candida albicans:identification of different isoleucine-valine, methionine, and arginine alleles by complementation. J. Bacren'ol. 151, 1247-1 252.
rnycoses 34, 75-83 ( 1991 )
83
14 Miller, L. & Berger, T. ( 1 985) Bacterial identification by gas chromatography of whole cell fatty acids. Hewlett Packard Application Note 228-41. Palo Alto, CA: Hewlett Packard co. 15 Bard, M., Albrecht, M. R., Gupta, N., Guynn, C. J. & Stillwell, W. (1988) Geraniol interferes with membrane functions in strains of Candida and Saccharomyces. Lipids 23, 534538. 16 Georgopapadakou, N. H., Dix, B. A., Smith, S. A., Freudenberger, J. & Funke, P. T. (1987) Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candica albicans. Antimicrob. Agents Chemother. 31, 46-51. 17 Lees, N. D., Lofton, S. L., Woods, R. A. & Bard, M. (1980) Effects of vaned energy source and detergent on the growth of sterol mutants of Saccharomyces cerevisiae. J. Gen. Microbiol. 118, 209-214. 18 Thomas, A. H. (1986) Suggested mechanisms for the antimycotic activity of the polyene antibiotics and the N-substituted imidazoles.J. Antimicrob. Chemother. 17, 269-279. 19 Subden, R. E., Safe, L., Moms, D. C., Brown, R. G. & Safe, S. (1977) Eburicol, lichesterol, ergosterol, and obtusifoliol from polyene antibiotic-resistant mutants of Candida albicans. Can.J. Microbiol. 23, 751-754. 20 Parks, L. W. (1978) Metabolism of sterols in yeast. CRC Crit. Rev. Microbiol. 6, 301-341. 21 Woods, R. A., Bard, M., Gardner, I. E. & Molzhan, S. W. (1974) Studies on the accumulation of ergosterol and 24(28)dehydroergosterol in 3 strains of Saccharomyces cerevisiae. Microbios 1 0 4 73-80. 22 Lees, N. D., Kleinhans, F. W., Broughton, M. C., Pennington, D. E., Ricker, V. A. & Bard, M. (1989) Membrane fluidity alterations in a cytochrome P450-deficient mutant of Candida albicans. Steroids 53, 567-578. 23 Lees, N. D., Bard, M., Kemple, M. D., Haak, R. A. & Kleinhans, F. W. (1979) ESR determination of membrane order parameter in yeast sterol mutants. Biochim. Biophys. Acta 553, 469-475. 24 Bard, M., Lees, N. D., Burrows, L. S., Kleinhans, F. W. (1978) Differencesin crystal violet uptake and cation-induced death among yeast sterol mutants. J. Bacteriol. 135, 11461148. 25 Lees, N. D., Broughton, M. C., Sanglard, D. & Bard, M. (1990) h o l e susceptibility and hyphal formation in a cytochrome P-450-deficient mutant of Candida albicans. Antimicrob. Agents Chemother. -83 1-836.
