Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 1998, p. 1581-1586, Vol. 42, No. 7
Bayer AG, Institut für Antiinfektiva
Forschung, D-42096 Wuppertal, Germany
Received 14 January 1998/Returned for modification 5 March
1998/Accepted 3 May 1998
BAY 10-8888, a cyclic Drug-resistant mutants of
susceptible fungi are important tools for unraveling the mode of action
of antifungals and for the characterization of possible resistance
mechanisms (2, 6, 7, 9, 12-14, 18, 20, 22, 23). For
example, resistant Saccharomyces cerevisiae mutants were
used to clone a subunit of the target protein for echinocandins, the
BAY 10-8888 is a synthetic derivative of the naturally occurring
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Decreased Accumulation or Increased Isoleucyl-tRNA Synthetase
Activity Confers Resistance to the Cyclic
-Amino Acid BAY
10-8888 in Candida albicans and Candida
tropicalis
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-amino acid, exerts its antifungal
activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have
selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a
structurally related -amino acid, while sensitivities to
5-fluorocytosine and fluconazole remained unchanged in all mutants. All
except two in vitro-resistant mutants were pathogenic in a murine
candidiasis model, and BAY 10-8888 failed to cure the infection.
Furthermore, we have characterized BAY 10-8888 transport and
isoleucyl-tRNA synthetase activity in several Candida
tropicalis strains which showed MICs higher than those of other
Candida strains. An analysis of the C. tropicalis strains
revealed that intracellular concentrations of BAY 10-8888 were in the
millimolar range, comparable to those for C. albicans.
However, these isolates expressed isoleucyl-tRNA synthetase activities
about fourfold higher than those for C. albicans. To test
the possibility of resistance modeling, we determined the correlations
between the intracellular concentration of BAY 10-8888, the specific
activity of isoleucyl-tRNA synthetase, the number of free, i.e.,
noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth,
assuming a linear relation. We found significant correlations between
growth and the intracellular concentration of BAY 10-8888 and between
growth and the number of free isoleucyl-tRNA synthetase molecules/cell,
but not between growth and the specific activity of isoleucyl-tRNA
synthetase.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-glucan synthase (6), and the gene encoding the target
for aureobasidin (12, 13, 23). Characterization of a
Candida albicans mutant resistant to nikkomycin
suggested that its uptake by a dipeptide permease is an important step
for nikkomycin action (22). Furthermore, 5-fluorocytosine-resistant clinical isolates of C. albicans
displayed a low level of activity of UMP pyrophosphorylase, an enzyme
which is part of the biochemical pathway converting 5-fluorocytosine into a DNA and RNA synthesis inhibitor (34).
-amino acid cispentacin (15, 17, 24, 25) with potent anti-Candida activity (4). The structures of BAY
10-8888 and cispentacin are shown in Fig.
1. Recently, we have unraveled the mode
of action of BAY 10-8888. BAY 10-8888 is accumulated about 200-fold by
Candida spp. and S. cerevisiae. Inside the cell,
BAY 10-8888 inhibits isoleucyl-tRNA synthetase, which results in the inhibition of protein biosynthesis and cell growth (37).
View larger version (6K):
[in a new window]
FIG. 1.
Structures of BAY 10-8888 [(-)-(1R,2S)-2-amino-4-methylene-cyclopentane-1-carboxylic acid] and
cispentacin [(-)-(1R,2S)-2-amino-cyclopentane-1-carboxylic acid]).
The characterization of BAY 10-8888-resistant C. albicans mutants, presented here, supports our current view of its mode of action. Furthermore, our studies explain the reduced sensitivities of some Candida tropicalis isolates.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Radiolabelled compounds. [14C]BAY 10-8888 (12 mCi/mmol) was provided by M. Radtke, Institute for Pharmacokinetics, Bayer AG. The radiochemical purity was >96%.
Materials.
The -amino acids cispentacin and BAY 10-8888 and fluconazole were synthesized at Bayer AG. 5-Fluorocytosine,
-chloro-DL-alanine, DL-thiaisoleucine,
cycloleucine, and cycloheximide were obtained from Sigma, Deisenhofen,
Germany.
Organisms. All C. albicans and C. tropicalis strains represent clinical isolates from various sources and are maintained in the Bayer strain collection mycology (BSM). The following yeasts were used: C. albicans BSMY 212 (ATCC 200498), C. tropicalis BSMY 601, C. tropicalis BSMY 605, C. tropicalis BSMY 610, C. tropicalis BSMY 611, C. tropicalis BSMY 612, C. tropicalis BSMY 616, and C. tropicalis BSMY 620. Strains were maintained and grown as described previously (37). C. albicans CAI4 was a gift from W. Fonzi (8).
Mutant selection. To select for C. albicans mutants resistant to BAY 10-8888, 108 log-phase C. albicans cells grown in YNG medium (0.67% Bacto yeast nitrogen base [Difco], 1.0% glucose [pH 7.0]) at 30°C were plated onto YNG medium-agar plates (1.5% agarose [Serva, Mannheim, Germany]) containing 100 or 500 µg of BAY 10-8888 per ml. After incubation for 48 h at 37°C the number of resistant colonies was determined. Susceptibility testing was performed as described previously with YNG medium (see above) (37). MIC90 was defined as the lowest compound concentration at which no visible growth occurred. Methods for measuring BAY 10-8888 uptake and isoleucyl-tRNA synthetase activity have been described previously (37).
Generation of resistant mutants by adaptation. C. albicans cells were inoculated at a cell density of 105/ml into YNG medium (10 ml) containing 1 µg of BAY 10-8888 per ml. Cells were incubated at 37°C with shaking until visible growth occurred. Then, 200 µl was transferred to fresh YNG medium containing 2 µg of BAY 10-8888 per ml, and incubation was continued at 37°C. By successive twofold increases in concentration over a period of about 1 month mutants resistant to 640 and 1,280 µg of BAY 10-8888 per ml were obtained. Clones were isolated by streaking out an aliquot on YNG-agar plates containing appropriate concentrations of BAY 10-8888.
In vivo pathogenicity and sensitivity. We used the mouse candidiasis model described in reference 26 to test C. albicans mutants for pathogenicity and BAY 10-8888 sensitivity. Briefly, mutant yeast variants were grown for 24 h at 28°C in Nervina Agar (0.5% [vol/vol] glycerol, 0.5% Bacto Peptone, 0.5% sodium chloride, 4% malt extract, 2% Bacto Agar; pH 7.0) tubes and rinsed off with phosphate-buffered saline (PBS). Mice were infected by injection of 106 cells in 0.2 ml of PBS into the caudal vein. Mice were treated orally (p.o.) with 10 mg of BAY 10-8888 per kg of body weight twice daily for 4 days starting immediately after infection. Nontreated controls were included. Survival was monitored for 7 days. Mice severely ill on day 7 were recorded as dead. Survival curves were calculated according to the Kaplan-Meier method with the Prism program (GraphPad Software Inc., San Diego, Calif.) for microcomputers and compared by the log rank test. A P value <0.05 was considered significant.
Calculation of the number of tRNA-synthetase molecules.
The
number of isoleucyl-tRNA synthetase molecules per cell was calculated
based on data from a procedure for the purification of isoleucyl-tRNA
synthetase from baker's yeast (32). Van der Haar isolated
160 A280 units (which is equivalent to 160 mg)
of isoleucyl-tRNA synthetase from 6,000 g of baker's yeast. Assuming a
wet weight of 60 pg/cell (30), this corresponds to
1014 cells. The purity of the isoleucyl-tRNA synthetase
preparation was 20 to 60%, and the yield (based on total activity) was
70.5%. The amount of isoleucyl-tRNA synthetase per cell can be
calculated as follows: 0.16 g × 0.2 × 1.41/1014
cells = 4.5 × 10
16 g/cell and 0.16 g × 0.6 × 1.41/1014 cells = 1.35 × 1015 g/cell, assuming 20 and 60% purity, respectively.
The molecular weight of isoleucyl-tRNA synthetase from S. cerevisiae is 124,000 (3). Thus, the number of
isoleucyl-tRNA synthetase molecules per cell can be calculated as
follows: 4.5 × 1016 g/cell × 6.022 × 1023 molecules/mol/124,000 g/mol = 2,185 molecules/cell and 1.35 × 1015 g/cell × 6.022 × 1023 molecules/mol/124,000 g/mol = 6,556 molecules/cell, again assuming 20 and 60% purity, respectively. The
specific activity of isoleucyl-tRNA synthetase (0.11 to 0.13 U/mg of
protein) in crude cell extract of C. albicans BMSY 212 was
similar to the specific activity (0.12 U/mg of protein) in crude cell
extract of baker's yeast (32). Assuming that C. albicans and S. cerevisiae contain the same amount of
protein per cell, the number of isoleucyl-tRNA synthetase molecules per
cell expressed by C. albicans BMSY 212 under the growth
conditions used here is similar to the number we calculated for
S. cerevisiae. The concentration of free (i.e., not
inhibited by BAY 10-8888) isoleucyl-tRNA synthetase
[Efree] was calculated according to the
following equation: [Efree] = Ki × [Et]/[Et + I], which
was derived from the dissociation equation Ki = [E] × [I]/[EI].
[Et] is the total concentration of
isoleucyl-tRNA synthetase inside the cell. Ki is
the dissociation constant of BAY 10-8888 (37), and
[I] is the intracellular concentration of BAY 10-8888. [Et] was calculated by the equation
[Et] M = number of isoleucyl-tRNA synthetase molecules/cell/(6.022 × 1023
molecules/mol × 5.7 × 1014 liters/cell) where
5.7 × 1014 liters is the volume of a C. albicans BSMY 212 cell (37). Therefore, [Et] is 6.4 × 108 M for
2,200 molecules/cell and 2 × 107 M for 6,600 molecules/cell.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Generation and characterization of BAY 10-8888-resistant C. albicans mutants.
C. albicans mutants resistant in
vitro to BAY 10-8888 were selected by two methods. To isolate C. albicans mutants that reveal a high level of resistance,
susceptible C. albicans BSMY 212 cells were plated on
YNG-agarose containing 100 or 500 µg of BAY 10-8888 per ml. After
incubation for 48 h at 37°C, BAY 10-8888-resistant colonies
appeared at a frequency of 1.2 × 107 and 3.8 × 107, respectively. Several colonies were picked from
plates containing 100 µg of BAY 10-8888 per ml (mutants S13 to S17)
and 500 µg of BAY 10-8888 per ml (mutants S18 to S22) and tested for
their sensitivities to BAY 10-8888, the related -amino acid
cispentacin, and the antifungals 5-fluorocytosine and fluconazole
(Table 1). For mutants selected on 100 µg of BAY 10-8888 per ml the MIC90s for BAY 10-8888 were
increased 250- to 8,000-fold, while all mutants selected on agar plates
containing 500 µg of BAY 10-8888 per ml showed about an 8,000-fold
decrease in susceptibility to BAY 10-8888. Susceptibilities to
cispentacin decreased (MIC90s, 64 to 128 µg/ml), except
for that of mutant S15. Susceptibilities to 5-fluorocytosine and
fluconazole remained unchanged. Sensitivity to BAY 10-8888 did not
increase after repeated passage of the mutants in BAY 10-8888-free
medium, indicating that the resistant phenotype was stable. Mutants S13
to S22 grew at rates comparable to that of the wild type (data not
shown).
|
-chloro-DL-alanine and
DL-thiaisoleucine increased from 31 (level for wild type) to >1,000 µg/ml; the MIC90 of cycloleucine increased
from 25 to >100 µg/ml. In contrast, the sensitivity of this mutant
to cycloheximide remained unchanged. Mutants 640/1 and 1280/1 grew as
fast as the wild type, whereas mutant 640/2 grew more slowly (data not
shown).
To test whether resistance was due to alterations in the target
molecules of BAY 10-8888, we investigated BAY 10-8888 accumulation under steady-state conditions and isoleucyl-tRNA synthetase activities in selected mutants. As shown previously, BAY 10-8888 inhibits isoleucyl-tRNA synthetase activity after accumulation to millimolar concentrations within the cell (37). The results are shown
in Table 2. All mutants showed reduced
BAY 10-8888 uptake after incubation for 30 min compared to the wild
type. Except for mutant S15, BAY 10-8888 accumulation was below 10% of
that of the wild type. The MIC90 for mutant S15, which
showed the highest residual activity (14.1% of that of the wild type),
was lower than that for any other mutant (Table 2).
|
|
Characterization of BAY 10-8888-resistant C. tropicalis
strains.
We determined the accumulations of BAY 10-8888 and the
specific isoleucyl-tRNA synthetase activities for seven C. tropicalis isolates less sensitive to BAY 10-8888 (MIC90s, 
64 µg/ml) in vitro (Table
3). Relative to C. albicans
BSMY 212 all C. tropicalis isolates accumulated 52 to 82%
BAY 10-8888 per mg of protein after 30 min. The specific activities of
isoleucyl-tRNA synthetase ranged from about 2.5- to 4-fold higher
compared to that of C. albicans BSMY 212 (Table 3).
|
Prediction of growth in the presence of BAY 10-8888. As shown above several C. albicans mutants and C. tropicalis isolates, less sensitive to BAY 10-8888 in vitro, showed reduced active accumulation of BAY 10-8888 or increased isoleucyl-tRNA synthetase activity or both. We tested whether changes in one of these two parameters or in the parameter resulting from the combination of the two, namely, the number of noninhibited isoleucyl-tRNA synthetase molecules per cell, can be correlated with growth at 8 µg of BAY 10-8888 per ml (56 µM). Mutants 640/1, 640/2, and 1280/1 were not included, since they were selected to grow at very high concentrations of BAY 10-8888 and may, therefore, have multiple mutations.
To calculate the number of isoleucyl-tRNA synthetase molecules per cell we used data from the closely related baker's yeast S. cerevisiae, because the corresponding data are not available for C. albicans. The total number of isoleucyl-tRNA synthetase molecules (Et) was calculated to range between 2,200 and 6,600 molecules/cell. Then, by using the equations described in Materials and Methods the number of free isoleucyl-tRNA synthetase molecules, i.e., those not inhibited in the presence of 8 µg of BAY 10-8888 per ml in the medium, per cell was calculated (Table 4). Next, we calculated the correlation coefficients assuming a linear relation between growth and the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, and the number of free isoleucyl-tRNA synthetase molecules per cell. The correlation coefficients, r, were
0.74 (P = 0.004) for the
correlation between relative growth and the intracellular concentration
of BAY 10-8888 (Fig. 3A), 0.27 (P = 0.37) for the
correlation of the specific activity of isoleucyl-tRNA synthetase with
relative growth (Fig. 3B), and 0.75 (P = 0.0029) for
the correlation between relative growth and the number of free
isoleucyl-tRNA synthetase molecules per cell (Fig. 3C). This indicated
that there was no linear correlation between growth and the specific
activity of isoleucyl-tRNA synthetase, while weak linear correlation
was observed between growth and the intracellular concentration of BAY
10-8888 and between growth and the number of free isoleucyl-tRNA
synthetase molecules/cell. More detailed inspection of the plot shown
in Fig. 3A revealed that C. albicans CAI4 and C. tropicalis isolates 605 and 620 were outliers. While C. tropicalis isolates 605 and 620 remained outliers when the number
of free isoleucyl-tRNA synthetase molecules per cell versus growth was
plotted (Fig. 3C), C. albicans CAI4, due to the high level
of specific activity of isoleucyl-tRNA synthetase, was no longer an
outlier. This suggested that C. tropicalis isolate 620 was
resistant due to other mechanisms and that the sensitivity of isolate
605 cannot be explained by the relatively high number of free
isoleucyl-tRNA synthetase molecules.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
We have selected and characterized C. albicans mutants
resistant to BAY 10-8888, an antifungal cyclic -amino acid. Mutants spontaneously resistant to high concentrations of BAY 10-8888 showed
decreased accumulation of BAY 10-8888. This provides additional evidence that accumulation is a prerequisite for the in vitro antifungal activity of BAY 10-8888 (37). The mutants were
only partially cross-resistant to cispentacin. This supports the
finding that cispentacin is accumulated by carriers specific for other amino acids (5, 16). Except for two, all resistant mutants were still pathogenic for mice in an intravenous challenge model. BAY
10-8888 did not cure infections with these mutants, indicating that the
accumulation of BAY 10-8888 is also important for its in vivo mode of
action. More detailed functional characterization of the carrier
transporting BAY 10-8888 for in vivo efficacy, viability, and
pathogenicity involves the cloning of the carrier and the generation of
C. albicans knockout mutants (8, 10, 11). From
our results, we cannot predict whether the kind of mutation we
characterized here occurs under in vivo conditions and, if so, at what
frequency. The reduced uptake of BAY 10-8888 in resistant mutants could
also be due to the increased expression of efflux pumps as, for
example, observed for azole-resistant strains (1, 27-29).
Because of the unchanged sensitivities of all mutants to the azole
fluconazole, we can exclude resistance due to overexpression of the
multidrug resistance transporter CDR1, but not other export pumps
(28, 29).
Mutants 640/1 and 1280/1, stepwise selected to grow in medium containing 640 and 1,280 µg of BAY 10-8888 per ml, respectively, were cross-resistant to cispentacin as well as to other toxic amino acid analogs. One can speculate that multiple permeases are affected, as described, for example, for the S. cerevisiae SHR3 mutation (21). In addition to reduced BAY 10-8888 accumulation, 640/1 and 640/2 had elevated levels of isoleucyl-tRNA synthetase, the target enzyme of BAY 10-8888. This was not observed in mutants spontaneously resistant to high concentrations of BAY 10-8888 and suggests that the mutation frequency for accumulation is higher than those for the subsequent resistance mechanisms. Mutant 1280/1 seems to have additional resistance mechanisms, e.g., mutation of the target enzyme.
We also characterized seven C. tropicalis isolates, which showed reduced in vitro sensitivity to BAY 10-8888. Despite intracellular accumulation of BAY 10-8888 to concentrations in the millimolar range, these isolates grew in the presence of 8 µg of BAY 10-8888 per ml, a concentration that is fourfold higher than the MIC90 for C. albicans BSMY 212. Interestingly, all isolates showed a higher level of specific activity of isoleucyl-tRNA synthetase. Since we have shown that overexpression of endogenous isoleucyl-tRNA synthetase in S. cerevisiae decreases BAY 10-8888 sensitivity (37), this may be responsible for the decreased sensitivity.
C. albicans and other fungi possess many resistance mechanisms to antifungals (1, 9, 27-29, 31, 33, 35, 36), which cannot all be monitored at the same time for practical reasons. Furthermore, it is always difficult to decide whether an observed change in, for example, the expression of the gene encoding the target, a mutation, or an increased expression of an efflux pump is indeed responsible for the resistance phenotype or represents only an epiphenomenon. Therefore, we tested whether one of the observed characteristics of BAY 10-8888-resistant C. albicans mutants and C. tropicalis isolates, decreased accumulation of BAY 10-8888 or increased specific activity of isoleucyl-tRNA synthetase, or whether, as a result of both effects, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules per cell can be linearly correlated with growth at 8 µg of BAY 10-8888 per ml. Due to many assumptions and unknown variables the number of free isoleucyl-tRNA synthetase molecules per cell is more a rough estimate than an exact calculation. Nevertheless, it proved to be useful to explain resistance to BAY 10-8888 (see below). While the specific activity of isoleucyl-tRNA synthetase could not be correlated with growth, the intracellular concentration of BAY 10-8888 and the number of free isoleucyl-tRNA synthetase molecules per cell showed significant but weak correlation. An examination of the graphs shown in Fig. 3A and C revealed that the two outliers, C. tropicalis 605 and 620, are, most likely, sensitive and resistant, respectively, to BAY 10-8888 because of other mechanisms. In contrast, C. albicans CAI4 only represents an outlier when the intracellular concentration of BAY 10-8888 is correlated with growth, not if the number of free isoleucyl-tRNA synthetase molecules per cell is correlated with growth. This suggests that the number of free isoleucyl-tRNA synthetase molecules per cell is a better parameter to explain the sensitivity of this strain. The number of free isoleucyl-tRNA synthetase molecules per cell is also sufficient to explain the sensitivity or resistance of the remaining isolates. However, this needs to be further validated, as we tested only a limited number of C. albicans strains. Furthermore, from a physiological point of view, one has to take into account the fact that the linear correlation we assumed occurs only within certain limits. For example, for unlimited growth a certain number of active isoleucyl-tRNA synthetase molecules is required. Further increase in the molecule number will not increase growth rate, as other reactions or metabolites will become growth limiting. On the other hand, a minimal number of active isoleucyl-tRNA synthetase molecules may be required for any growth to occur.
|
The resistance mechanisms observed for BAY 10-8888 are different from
those for other antifungals. Resistance against 5-fluorocytosine can
result from mutation of any of the enzymes (cytosine permease, cytosine
deaminase, uridine 5'-monophosphate
pyrophosphate-phosphoribosyltransferase) involved in its conversion and
incorporation in RNA (31). For azoles several resistance
mechanisms have been described. These include overexpression or
mutation of the targets enzyme lanosterol 14
demethylase (19,
35, 36). The major resistance mechanism, however, is increased
expression of ABC transporters or major facilitator pumps (1,
27-29, 33, 35). None of the C. albicans mutants
showed any cross-resistance to fluconazole or 5-fluorocytosine, indicating that resistance mechanisms against BAY 10-8888 are different
from those against fluconazole and 5-fluorocytosine.
In summary, our data as well as our theoretical considerations fully
support the two-step mode of action for the antifungal -amino acid
BAY 10-8888, namely, inhibition of isoleucyl-tRNA synthetase after
active accumulation inside the cell.
| |
ACKNOWLEDGMENTS |
|---|
I thank W. Schönfeld for critically reading the manuscript and S. Badock and A. Ludwig for excellent technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Bayer Yakuhin Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-02, Japan. Phone: (81)774 75-2462. Fax: (81)774 75-2507. E-mail: karl.ziegelbauer.kz{at}bayer-ag.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Albertson, G. D., M. Niimi, R. D. Cannon, and H. F. Jenkinson. 1996. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob. Agents Chemother. 40:2835-2841[Abstract]. |
| 2. | Angiolella, L., C. Bromuro, N. Simonetti, and A. Cassone. 1992. Glucan synthesis and its inhibition by cilofungin in susceptible and resistant strains of Candida albicans. J. Met. Vet. Mycol. 30:369-376. |
| 3. | Bhanot, O. S., Z. Kucan, S. Aoyagi, F. C. Lee, and R. W. Chambers. 1974. Purification of tyrosine transfer RNA ligase, valine transfer RNA ligase, alanine transfer ligase and isoleucine transfer ligase from Saccharomyces-cerevisiae alpha S-288C. Methods Enzymol. 29:547-576. |
| 4. | Bremm, K., R. Endermann, F. Kunisch, M. Matzke, K.-G. Metzger, H. Militzer, J. Mittendorf, and M. Plempel. May 1993. EP patent 571,870. |
| 5. | Capobianco, J., D. Z. Zakula, M. L. Coen, and R. C. Goldman. 1993. Anti-Candida activity of cispentacin: the active transport of amino acid permeases and its possible mechanism of action. Biochem. Biophys. Res. Commun. 190:1037-1044[Medline]. |
| 6. |
Douglas, C. M.,
F. Foor,
J. A. Marrinan,
N. Morin,
J. B. Nielsen,
A. M. Dahl,
P. Mazur,
W. Baginsky,
W. L. Li,
M. El-Sherbeini,
J. A. Clemas,
S. M. Mandala,
B. R. Frommer, and M. B. Kurtz.
1994.
The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane-protein which is a subunit of 1,3-beta-D-glucan synthase.
Proc. Natl. Acad. Sci. USA
91:12907-12911 |
| 7. |
Douglas, C. M.,
J. A. Marrinan,
W. Li, and M. B. Kurtz.
1994.
A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3--D-glucan synthase.
J. Bacteriol.
176:5686-5696 |
| 8. | Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717-728[Abstract]. |
| 9. | Frost, D. J., M. Knapp, K. Brandt, A. Shadron, and R. C. Goldman. 1997. Characterization of a lipopeptide-resistant strain of Candida albicans. Can. J. Microbiol. 43:122-128[Medline]. |
| 10. |
Goldway, M.,
D. Teff,
R. Schmidt,
A. B. Oppenheim, and Y. Koltin.
1995.
Multidrug resistance in Candida albicans: disruption of the BENr gene.
Antimicrob. Agents Chemother.
39:422-426 |
| 11. |
Gow, N. A. R.,
P. W. Robbins,
J. W. Lester,
A. J. P. Brown,
W. A. Fonzi,
T. Chapman, and O. S. Kinsman.
1994.
A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.
Proc. Natl. Acad. Sci. USA
91:6216-6220 |
| 12. |
Hashidaokado, T.,
A. Ogawa,
M. Endo,
R. Yasumoto,
K. Takesako, and I. Kato.
1996.
AUR1, a novel gene conferring aureobasidin resistance on Saccharomyces cerevisiae a study of defective morphologies in AUR1P-depleted cells.
Mol. Gen. Genet.
251:236-244[Medline].
|
| 13. | Heidler, S. A., and J. A. Radding. 1995. The AUR1 gene in Saccharomyces cerevisiae encodes dominant resistance to the antifungal agent auroeobasidin A (LY295337). Antimicrob. Agents Chemother. 39:2765-2769[Abstract]. |
| 14. | Hitchcock, C. A., N. J. Russell, and B. K. Barrett. 1987. Sterols in Candida albicans mutants resistant to polyene or azole antifungals, and of a double mutant C. albicans 6.4. Crit. Rev. Microbiol. 15:111-115[Medline]. |
| 15. | Iwamoto, T., E. Tsujii, M. Ezaki, A. Fujie, S. Hashimoto, M. Okuhara, M. Kohsaka, and H. Imanaka. 1990. FR109615, a new antifungal antibiotic from Streptomyces setonii: taxonomy, fermentation, isolation, physico-chemical properties and biological activity. J. Antibiot. 43:1-7[Medline]. |
| 16. |
Jethwaney, D.,
M. Hofer,
R. K. Khaware, and R. Prasad.
1997.
Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin.
Microbiology
143:397-404 |
| 17. | Konishi, M., M. Nishio, K. Saitoh, T. Miyaki, T. Oki, and K. Kawaguchi. 1989. Cispentacin, a new antifungal antibiotic. I. Production, isolation, physico-chemical properties and structure. J. Antibiot. 42:1749-1755[Medline]. |
| 18. | Kurtz, M. B., G. Abruzzo, A. Flatlery, K. Bartizal, J. A. Marrinan, W. Li, J. Milligan, K. Nollstadt, and C. M. Douglas. 1996. Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies. Infect. Immun. 64:3244-3251[Abstract]. |
| 19. |
Lamb, D. C.,
D. E. Kelly,
W. H. Schunck,
A. Z. Shyadehi,
M. Akhtar,
D. J. Lowe,
B. C. Baldwin, and S. L. Kelly.
1997.
The mutation T315A in Candida albicans sterol 14 alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.
J. Biol. Chem.
272:5682-5688 |
| 20. |
Lees, N. D.,
M. C. Broughton,
D. Sanglard, and M. Bard.
1990.
Azole susceptibility and hyphal formation in a cytochrome P-450-deficient mutant of Candida albicans.
Antimicrob. Agents Chemother.
34:831-836 |
| 21. | Ljungdahl, P. O., C. Gimeno, C. A. Styles, and G. R. Fink. 1992. A novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast. Cell 71:463-478[Medline]. |
| 22. |
McCarthy, P. J.,
P. F. Troke, and K. Gull.
1985.
Mechanism of action of nikkomycin and the peptide transport system of Candida albicans.
J. Gen. Microbiol.
131:775-780 |
| 23. |
Nagiec, M. M.,
E. E. Nagiec,
J. A. Baltisberger,
G. B. Wells,
R. L. Lester, and R. C. Dickson.
1997.
Sphingolipid synthesis as a target for antifungal drugscomplementation of the inositol phosphoceramide synthase defect in a strain of Saccharomyces cerevisiae by the AUR1 gene.
J. Biol. Chem.
272:9809-9817 |
| 24. | Ohki, H., Y. Inamoto, K. Kawabata, T. Kamimura, and K. Sakane. 1991. Synthesis and antifungal activity of FR109615 analogs. J. Antibiot. 44:546-549[Medline]. |
| 25. | Oki, T., M. Hirano, K. Tomatsu, K. Numata, and H. Kamei. 1989. Cispentacin, a new antifungal antibiotic. II. In vitro and in vivo antifungal activities. J. Antibiot. 42:1756-1762[Medline]. |
| 26. |
Plempel, M.
1984.
Antimycotic activity of Bay N 7133 in animal experiments.
J. Antimicrob. Chemother.
13:447-463 |
| 27. |
Sanglard, D.,
F. Ischer,
M. Monod, and J. Bille.
1997.
Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene.
Microbiology
143:405-416 |
| 28. | Sanglard, D., F. Ischer, M. Monod, and J. Bille. 1996. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob. Agents Chemother. 40:2300-2305[Abstract]. |
| 29. | Sanglard, D., K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille. 1995. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother. 39:2378-2386[Abstract]. |
| 30. | Sherman, F. 1991. Getting started with yeast. Methods Enzymol. 194:3-21[Medline]. |
| 31. | Vanden Bosche, H., P. Marchial, and F. C. Odds. 1994. Molecular mechanisms of drug resistance in fungi. Trends Microbiol. 2:393-400[Medline]. |
| 32. | Van der Haar, F. 1979. Purification of aminoacyl-tRNA synthetases. Methods Enzymol. 59:257-267[Medline]. |
| 33. | Walsh, T. J., M. Kasai, A. Franscesconi, D. Landsman, and S. J. Chanock. 1997. New evidence that Candida albicans possesses additional ATP-binding cassette MDR-like genes: implications for antifungal azole resistance. J. Med. Vet. Mycol. 35:133-137[Medline]. |
| 34. |
Whelan, W. L., and D. Kerridge.
1984.
Decreased activity of UMP pyrophosphorylase associated with resistance to 5-fluorocytosine in Candida albicans.
Antimicrob. Agents Chemother.
26:570-574 |
| 35. | White, T. C. 1997. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 41:1482-1487[Abstract]. |
| 36. |
White, T. C.
1997.
The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14 demethylase in Candida albicans.
Antimicrob. Agents Chemother.
41:1488-1494[Abstract].
|
| 37. | Ziegelbauer, K., P. Babczinski, and W. Schönfeld. Submitted for publication. |
Antimicrobial Agents and Chemotherapy, July 1998, p. 1581-1586, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hasenoehrl, A., Galic, T., Ergovic, G., Marsic, N., Skerlev, M., Mittendorf, J., Geschke, U., Schmidt, A., Schoenfeld, W.
(2006). In Vitro Activity and In Vivo Efficacy of Icofungipen (PLD-118), a Novel Oral Antifungal Agent, against the Pathogenic Yeast Candida albicans.. Antimicrob. Agents Chemother.
50: 3011-3018
[Abstract] [Full Text] -
Petraitiene, R., Petraitis, V., Kelaher, A. M., Sarafandi, A. A., Mickiene, D., Groll, A. H., Sein, T., Bacher, J., Walsh, T. J.
(2005). Efficacy, Plasma Pharmacokinetics, and Safety of Icofungipen, an Inhibitor of Candida Isoleucyl-tRNA Synthetase, in Treatment of Experimental Disseminated Candidiasis in Persistently Neutropenic Rabbits. Antimicrob. Agents Chemother.
49: 2084-2092
[Abstract] [Full Text] -
Petraitis, V., Petraitiene, R., Kelaher, A. M., Sarafandi, A. A., Sein, T., Mickiene, D., Bacher, J., Groll, A. H., Walsh, T. J.
(2004). Efficacy of PLD-118, a Novel Inhibitor of Candida Isoleucyl-tRNA Synthetase, against Experimental Oropharyngeal and Esophageal Candidiasis Caused by Fluconazole-Resistant C. albicans. Antimicrob. Agents Chemother.
48: 3959-3967
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»