ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2004, p. 16001613 0066-4804/04/$08.000 DOI: 10.1128/AAC.48.5.16001613.

2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 5

Functional Genomic Analysis of Fluconazole Susceptibility in the Pathogenic Yeast Candida glabrata: Roles of Calcium Signaling and Mitochondria
Rupinder Kaur, Irene Castan o, and Brendan P. Cormack*
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received 10 September 2003/Returned for modication 9 October 2003/Accepted 2 February 2004

The pathogenic yeast Candida glabrata exhibits innate resistance to uconazole, the most commonly used antifungal agent. By screening a library of 9,216 random insertion mutants, we identied a set of 27 genes which upon mutation, confer altered uconazole susceptibility in C. glabrata. Homologues of three of these genes have been implicated in azole and/or drug resistance in Saccharomyces cerevisiae: two of these belong to the family of ABC transporters (PDR5 and PDR16), and one is involved in retrograde signaling from mitochondria to nucleus (RTG2). The remaining 24 genes are involved in diverse cellular functions, including ribosomal biogenesis and mitochondrial function, activation of RNA polymerase II transcription, nuclear ubiquitin ligase function, cell wall biosynthesis, and calcium homeostasis. We characterized two sets of mutants in more detail. Strains defective in a putative plasma membrane calcium channel (Cch1-Mid1) were modestly more susceptible to uconazole but showed a signicant loss of viability upon prolonged uconazole exposure, suggesting that calcium signaling is required for survival of azole stress in C. glabrata. These mutants were defective in calcium uptake in response to uconazole exposure. The combined results suggest that, in the absence of Ca2 signaling, uconazole has a fungicidal rather than a fungistatic effect on C. glabrata. The second set of mutants characterized in detail were defective in mitochondrial assembly and organization, and these exhibited very high levels of uconazole resistance. Further analysis of these mutants indicated that in C. glabrata a mechanism exists for reversible loss of mitochondrial function that does not involve loss of mitochondrial genome and that C. glabrata can switch between states of mitochondrial competence and incompetence in response to uconazole exposure. The incidence of opportunistic fungal infections in the immunocompromised patient population has increased signicantly over the last two decades (62, 65). Candida species account for the majority of fungal infections, and these are primarily due to Candida albicans (47, 65). Coinciding with the introduction and widespread prophylactic use of the triazole antifungal uconazole, there has been a notable increase in the relative proportion of infections due to non-C. albicans Candida species: from 1989 to 1999, the incidence of nosocomial C. albicans bloodstream infections dropped fourfold, whereas the incidence of non-C. albicans bloodstream infections in the same population remained strikingly constant (43, 65). Whereas the rate of infections due to all non-C. albicans species remained constant, there was a statistically signicant increase in the rate of infections due to Candida glabrata, which is now the second most common cause of bloodstream and mucosal infections in the United States (6, 48, 65, 69). Perhaps related to its increasing prevalence, C. glabrata is innately resistant to uconazole (49) and indeed is often isolated as a replacement species in patients receiving uconazole treatment (47, 48, 69). The triazole class of compounds are key antifungal agents, with uconazole being the most commonly used antifungal agent to treat Candida infections due to its high efciency, low toxicity, and oral availability (18). The triazoles target cyto* Corresponding author. Mailing address: PCTB 522, Johns Hopkins University School of Medicine, Department of Molecular Biology and Genetics, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 614-4923. Fax: (410) 502-6718. E-mail: bcormack@bs.jhmi.edu. 1600

chrome P-450-dependent C14 lanosterol demethylase (encoded by ERG11), an essential enzyme in the ergosterol biosynthesis in fungi (24, 31, 72). The nitrogen atoms in the azole ring bind to the heme group in the active site of the enzyme, thereby blocking the binding of the normal substrate (72). The efcacy of triazole compounds has been proposed to be due to both the depletion of ergosterol in the plasma membrane and the intracellular accumulation of toxic 14-methylated sterol intermediates, ultimately leading to growth arrest but not cell death (26, 29, 31, 58). The molecular mechanism of azole resistance has been extensively studied both in the clinical isolates of C. albicans, as well as in the model yeast organism, Saccharomyces cerevisiae. The principal biochemical mechanisms associated with azole resistance include drug target (ERG11) amplication, structural modications in the Erg11p enzyme, mutations in other ergosterol biosynthesis genes that result in the accumulation of alternative nontoxic sterols, and reduced drug uptake and enhanced drug efux due to the overexpression of membraneassociated multidrug resistance transporters (25, 27, 30, 40, 51, 61, 66, 70, 71). Recently, studies of global gene expression analysis in the azole-resistant strains of C. albicans or in S. cerevisiae strains exposed transiently to azoles demonstrated upregulation of various genes previously implicated in azole resistance (3, 10, 54, 55). In addition, these studies uncovered new genes involved in oxidative stress response that seem to be coordinately regulated with the multidrug resistance transporter genes, CDR1, CDR2, and CaMDR1 (55).

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In addition to the moderate resistance to uconazole displayed by clinical isolates, C. glabrata, like C. albicans, can become even more resistant after selection in the presence of drug. The major described mechanisms of acquired azole resistance in C. glabrata clinical isolates include transcriptional upregulation of the CgERG11 gene and increased drug efux due to the upregulation of CDR1 and CDR2 (40, 57, 67). In addition, a high frequency of acquired azole resistance in vitro in C. glabrata has been linked to a loss of mitochondrial function (11, 56). Although the precise molecular mechanism underlying uconazole resistance and dysfunctional mitochondria in C. glabrata is not well understood, a likely mechanism involves the over expression in petites of the multidrug resistance pumps, Cdr1p and Cdr2p (56). This is consistent with reports for S. cerevisiae which demonstrate that dysfunctional mitochondria send a signal to the nucleus via a retrograde signaling pathway that leads to the over expression of many genes, including the pleiotropic drug resistance transcriptional factors, PDR1 and PDR3 (21, 73). The activation of PDR3 further leads to transcriptional induction of genes encoding multidrug resistance pumps, including PDR5 (an S. cerevisiae homolog of Candida CDR1) (2, 21, 51, 73). It has also been suggested that functional mitochondria increase azole susceptibility due to the conversion, under conditions of Erg11p inhibition, of nontoxic sterol intermediates to toxic sterol compounds (25, 29). Despite a clear association in vitro between mitochondrial dysfunction and uconazole resistance, the importance of mitochondrial dysfunction in clinical isolates is not clear. As mentioned above, the azoles are fungistatic rather than fungicidal, and this lack of fungicidal activity could limit the efcacy of this class of compounds in long-term treatment regimes, even providing a fertile selective environment for the emergence of drug resistant strains. Recently, the immunosuppressive drugs FK506 and cyclosporine have been shown to act synergistically with azole compounds, converting azoles from fungistatic to fungicidal drugs (9, 14, 37) without signicantly altering the susceptibility (50% MIC [MIC50]) to the drug per se. Both FK506 and cyclosporine act via the inhibition of a highly conserved Ca2/calmodulin-dependent serine/threonine protein phosphatase, calcineurin (34), a key component of calcium signaling in yeast (22). Calcineurin is required to survive membrane stress in yeast (7, 9) and is required for tolerance of C. albicans to antifungal compounds (5, 59). Calcium signaling in yeast controls diverse cellular processes, including pheromone arrest, morphogenesis, and cell cycle regulation. It is believed that membrane perturbation by azole treatment leads to an inux of extracellular calcium (Ca2) through the plasma membrane or its release from intracellular stores into the cytosol (14). Intracellular Ca2 is compartmentalized in different organelles, including the endoplasmic reticulum (ER), mitochondria, and vacuole. Cells have evolved many regulatory mechanisms to maintain the cytosolic concentration of Ca2 required for intracellular signaling by coordinating the efux and inux of Ca2 from the extracellular environment (50). One of the mechanisms of Ca2 inux through the plasma membrane is referred to as capacitative Ca2 entry (46) and is mediated by a high-afnity Ca2 channel composed of Cch1p and Mid1p (15, 23, 44). Cch1p is homologous to the catalytic subunit of voltage-gated

Ca2 channels in mammalian cells, and Mid1p is an N-glycosylated, integral plasma membrane protein that functions as the regulatory subunit of this channel (20). Both Cch1p and Mid1p proteins have been shown to be necessary for Ca2 inux and survival of ER stress in S. cerevisiae (7, 15, 23, 36, 44). We have utilized here a functional genomic approach to identify the major factors responsible for the limited uconazole susceptibility of C. glabrata by identifying mutants that display an altered uconazole susceptibility. One important class of mutants that led to high-level uconazole resistance resulted from the disruption of genes important in mitochondrial function. We report here that high-frequency (mitochondrion-related) acquired uconazole resistance is always associated with the loss of the mitochondrial function but, in many isolates, not with the loss of the mitochondrial genome. In such isolates, the mitochondrion-incompetent uconazole resistance phenotype is in fact a revertible phenotype. In addition, the rate at which mitochondrial incompetence can be reacquired in these isolates is much higher than the expected mutation rate, suggesting that C. glabrata can switch between states of mitochondrial competence and incompetence in response to azole exposure. We also found, as has been previously reported for C. glabrata (40, 57), that multidrug transporters have a signicant role in reducing uconazole susceptibility. In addition, we identied two mutants implicating calcium signaling with uconazole susceptibility in C. glabrata. Our study demonstrates for the rst time that C. glabrata mutants defective in the Cch1/Mid1p Ca2 channel lose viability upon prolonged uconazole exposure, suggesting that calcium signaling is required to survive uconazole stress in C. glabrata.
MATERIALS AND METHODS Media and chemicals. Yeast media were prepared as described previously (63) and 2% agar was added for plates. Yeast extract-peptone-dextrose (YPD) medium contained 10 g of yeast extract/liter and 20 g of peptone/liter supplemented with 2% glucose. Yeast extract-peptone-glycerol (YPG) medium contained 10 g of yeast extract/liter, 20 g of peptone/liter, and 3% glycerol. Selective medium contains yeast nitrogen base supplemented with 0.6% Casamino Acids. Bacterial media was prepared as described previously (1) and 1.5% agar was used for plates. Luria-Bertani (LB) medium contained yeast extract 5 of g/liter, Bacto Peptone (10 g/liter), and NaCl 10 of g/liter supplemented with 30 g of kanamycin/ml (Km30) where needed. Phosphate-buffered saline (PBS) is composed of 8 of g of NaCl/liter, 0.2 g of KCl/liter, 1.65 g of Na2HPO47H2O/liter, and 0.2 g of KH2PO4/liter. YPD plates containing various concentrations of uconazole were prepared by adding appropriate volume of uconazole (Diucan-Pzer) from a stock solution of 2 mg/ml. Strains and growth conditions. Escherichia coli strain BW23473 (lac-169 robA1 creC510 hsdR514 uidA::pir endA recA1) (39) was used to rescue the plasmids from C. glabrata insertion mutants. C. glabrata strains BG2 and BG14 (ura3::Tn903Neor) were used for all of the experiments described. Yeast cells were routinely grown at 30C on standard S. cerevisiae media (63), and E. coli strain was grown at 37C on yeast extract-tryptone media. Tn7 mutagenesis. In vitro mutagenesis of plasmids with a modied Tn7 transposon was carried out as described previously (8). Briey, we mutagenized fosmid DNAs carrying C. glabrata genomic DNA by using Tn7 in vitro. Pools of insertions in a given fosmid were recovered in Escherichia coli, and mutagenized fosmid DNA was prepared. Genomic DNA was released from the fosmid backbone by restriction endonuclease digestion and introduced into the genome of C. glabrata. The majority of insertions are double crossovers that replace the wild-type allele with a disrupted allele. A total of 10% of insertions are nonhomologous ectopic integrants. These are easily identied (see below) by PCR and were excluded from further analysis.

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mutant library represents only 20 to 25% of the genome, most fosmids analyzed here will not physically overlap. Therefore, the number of fosmids from which mutants with a phenotype were derived, rather than the number of mutants themselves, gives a good indication of the number of loci identied. This hypothesis was tested experimentally by rescuing multiple uconazole-susceptible or -resistant mutants from 10 different fosmids; the Tn7 insertions were always found in the same gene in all of the uconazole-susceptible or -resistant mutants derived from the same fosmid. Thus, for the analysis of uconazole-susceptible mutants, one representative mutant per fosmid was selected for further characterization. DAPI staining. To stain cells with DAPI (4,6-diamidino-2-phenylindole), 1 ml of overnight culture was taken and then washed once with PBS, and the cells were suspended in 1 ml of 4% p-formaldehyde. After incubation at room temperature for 2 h, the cells were washed thrice with PBS and resuspended in PBS. Then, 100 l of cell suspension was incubated with 1 l of DAPI (0.2 mg/ml) for 30 to 40 min, and the cells were visualized by uorescence microscopy. Trypan blue exclusion assay. Cells were grown in YPD medium at 30C overnight and inoculated to an optical density at 600 nm (OD600) of 0.01 in minimal medium containing different concentrations of uconazole. After 24 h of incubation, 1 ml of cells was harvested by centrifugation, washed with PBS, resuspended in PBS, and stained with 0.4% trypan blue for 5 to 10 min. A minimum of 300 cells (viable [unstained] and dead [stained] cells) was counted microscopically for each data set. Viable count assay. Cells were grown in YPD medium at 30C overnight and inoculated to an OD600 of 0.01 in minimal medium containing different concentrations of uconazole with or without FK506. After 24 h of incubation, 100 l of cell suspension was taken and diluted in PBS, and the total number of cells was counted with hemocytometer. Appropriate dilutions were plated on YPD and YPG plates to assess the number of viable cells. CFU on YPG plates were counted after 2 days of incubation at 30C as the nal readout, although essentially the same number of CFU was obtained on both YPG and YPD plates if small colonies (petites, as tested by their inability to grow on glycerol) from YPD plates were excluded from the nal viable cell counts. 45 Ca2 accumulation assay. The total intracellular 45Ca2 levels were measured as described previously (7). Briey, cells were grown in YPD to log phase, harvested, washed, and suspended in YPD containing 2 Ci of 45CaCl2/ml (ICN Biomedicals). Fluconazole and FK506 were added to a nal concentration of 64 and 2 g/ml, respectively. After the cells were grown at 30C for 4 h, cells were collected on GF/F lters (Whatman). The lters were washed thrice with buffer (10 mM CaCl2 and 5 mM HEPES-NaOH [pH 6.5]) and then dried at 85C for 1 to 2 h. The cellular accumulation of 45Ca2 was quantitated by liquid scintillation counting.

Yeast transformation. C. glabrata cells were transformed as described previously (16). Briey, cells were grown to early log phase in YPD, harvested, and washed with an equal volume of sterile water. The cells were then resuspended in a 1/100 volume of 100 mM lithium acetate, and 50-l aliquots were used for each transformation. To each tube containing 50 l of cell suspension, 240 l of 50% PEG (3500) was added, followed by 36 l of 1 M lithium acetate, 50 g of heat-denatured salmon sperm DNA, and 50 l of transforming DNA. Tubes containing transforming mix were incubated at 30C for 45 min, and 43 l of dimethyl sulfoxide (DMSO) was added before heat shock treatment at 42C for 15 min. The cells were centrifuged, resuspended in H2O, and plated on selective media. Screening of mutant library. Mutants were grown in 96-well plates at 30C for overnight. A 360 dilution from overnight cultures was made in PBS and 5 l of this cell suspension (about 5,000 cells) was spotted onto agar plates containing different concentrations of uconazole with a 96-pin replicator. Growth phenotype of mutants was scored after incubation at 30C for 2 days. Fluconazole susceptibility. Susceptibility of C. glabrata mutants to uconazole was tested by using the broth microdilution assay according to NCCLS protocol M27-A (42) with morpholinepropanesulfonic acid-buffered (pH 6.2) RPMI 1640 medium. Essentially, 104 cells were inoculated in RPMI 1640 medium supplemented with 0.5% ammonium sulfate. Endpoint readings were measured with a microplate reader after 24 h of growth with shaking at 35C. A uconazole concentration yielding 80% growth inhibition compared to the growth in drugfree medium was dened as the MIC at which 80% of the isolates tested are inhibited (MIC80). These assays were carried out in duplicate. Susceptibility of the C. glabrata mutants was also tested qualitatively by spotting serial dilutions of yeast cells onto YPD agar plates containing different concentrations of uconazole. The yeast strains were grown overnight at 30C in YPD liquid medium, cells were diluted to 2 108 cells/ml in PBS, and 5 l of the cell suspension and 10-fold serial dilutions of the yeast cells were spotted onto different plates, followed by incubation for 1 to 2 days at 30C. The uconazole breakpoint on YPD plates containing uconazole was considered to be the drug concentration at which no mitochondrion-competent individual colonies were observed. Mapping of Tn7 insertion. To map Tn7 insertion in C. glabrata mutants, Tn7 transposon from the disrupted locus was rescued along with the anking genomic fragment. Briey, genomic DNA was isolated from each mutant, digested with either MfeI or SpeI enzyme, recircularized, and transformed into BW23473 cells by electroporation as described previously (8). The transformants were selected on LB plates containing kanamycin (30 g/ml), and DNA was prepared by using a Qiagen Miniprep kit. The resulting circular plasmid contains the Tn7 transposon anked on either side by the gene it disrupts in the genome of C. glabrata mutant. Sequence analysis of the rescued plasmids with outward primers from Tn7 right and left ends revealed the identity of the disrupted genes. The sequence obtained from each plasmid (1.2 kb) was blasted against the S. cerevisiae genome database (http://seq.yeastgenome.org/cgi-bin/SGD/nph-blast2sgd) and the C. glabrata Genolevures database (http://cbi.labri.fr/Genolevures/advanced blast.php3), and the mutant and open reading frame names were designated accordingly. Regeneration of mutants. Genomic DNA was isolated from mutant strain and digested with either MfeI or SpeI enzyme, recircularized, and transformed into BW23473 cells by electroporation as described previously (8). The transformants were selected on LB plates containing kanamycin (30 g/ml), and DNA was prepared by Qiagen Miniprep kit. The rescued plasmid was sequenced with the outward primers from the Tn7 right and left ends. Using the sequence information from each end of the transposon, primers were designed corresponding to the sequence of the rescued genomic loci, and PCR was carried out on genomic DNA from mutants. The presence of a large band corresponding to the locus with a Tn7 insertion indicated disruption of the locus. The presence of both a small wild-type band and a large band carrying Tn7 in these mutants indicated that the transposon had integrated into the genome by nonhomologous integration. Such nonhomologous integration mutants were not further analyzed (for a detailed description of the mutagenesis and analysis of insertion mutants, see reference 8). The original Tn7 insertion mutation was regenerated by transforming MfeI- or SpeI-digested rescued plasmid into C. glabrata BG14 strain. Transformants were colony puried on synthetic minimal medium plates lacking uracil; for all insertions, disruption of the wild-type locus was conrmed by PCR as described above, and uconazole susceptibility was scored by spotting cells on plates containing uconazole. Consideration of multiple mutants derived from a single fosmid. The expectation is that if a fosmid contains a gene that can mutate to uconazole susceptibility, then in a pool of 100 transformants derived from that particular fosmid there will likely be multiple uconazole-susceptible mutants. These multiple mutants most likely represent independent insertions in the same gene. Since our

RESULTS Construction and screening of a C. glabrata mutant library. A library of 9,216 random insertion mutants covering 25% of the genome of C. glabrata was generated through homologous recombination of Tn7-mutagenized, linearized C. glabrata genomic fragments (8). This mutant library was screened individually in a 96-well format for strains that displayed altered susceptibility to uconazole. The C. glabrata wild-type strain BG2 showed robust growth on agar plates containing 8 g of uconazole/ml, partial inhibition of growth on agar plates containing 16 g of uconazole/ml, and essentially no growth on plates containing uconazole at a concentration of 32 g/ml (Fig. 1). The mutant library was screened for two classes of mutants: hypersusceptible mutants that showed growth defects on plates with 8 g of uconazole/ml and resistant mutants that displayed robust growth in the presence of 32 g of uconazole/ml. The screen yielded 73 hypersusceptible mutants, corresponding to 19 genomic loci, that showed an increased susceptibility to uconazole (8 g/ml). Of these 73 mutants, 14 were also unable to grow at 4 g of uconazole/ml, whereas the rest showed growth phenotypes only at a concentration of 8 g of uconazole/ml (Fig. 1A and data not shown). Two were found

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FIG. 1. Fluconazole susceptibility proles of C. glabrata mutants. Wild-type and mutant strains were grown in YPD for overnight at 30C, the OD600 was normalized, and 5 l of 10-fold serial dilutions were spotted onto YPD plates with or without various concentrations of uconazole (4 to 256 g/ml) as indicated. Plates were photographed after 2 days of growth at 30C. (A) Growth proles of uconazole-susceptible mutants on different concentrations of uconazole. cdr1-P mutant has a Tn7 insertion in the promoter region of CDR1 and displays susceptibility to 8 g of uconazole/ml compared to the cdr1 mutant (Tn7 insertion in the open reading frame), which is hypersusceptible even to 4 g of uconazole/ml. (B) Growth proles of uconazole-resistant mutants in the presence of various concentrations of uconazole.

to be due to nonhomologous integration events (see Materials and Methods) and were not further analyzed; the remaining 17 were analyzed as described in next section. The screen for uconazole-resistant mutants yielded 68 mutants (corresponding to 26 loci) that displayed robust growth in the presence of 32 g of uconazole/ml (data not shown). A total of 39 mutants (representing 13 loci) showed resistance to higher levels of uconazole (64 g/ml) and were selected for further analysis (Fig. 1B and data not shown). Of these, two were found to be due to nonhomologous integration events (see Materials and Methods) and were not further analyzed. Within the class of mutants resistant to 64 g of ucon-

azole/ml, 22 of the mutants (all derived from one mutagenized fosmid) were found to be in the same locus, a 2-kb noncoding region. The sequence of this intergenic fragment showed no homology with or similarity to known sequences in basic local alignment search tool (BLAST) searches (data not shown), and these mutants have not been further analyzed here. The remaining 15 mutants were tested for resistance to uconazole concentrations ranging from 128 to 512 g/ml. Of the 15 mutants (representing 10 loci) in total, 6 mutants (representing four loci) were resistant to 64 g of uconazole/ml, 5 mutants (representing three loci) were resistant to 128 g of uconazole/ml, and 4 mutants (representing three loci) were resis-

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Mutants identied in a screen for uconazole sensitivity in C. glabrata

Wild type or mutant

Fluconazole MIC80 (g/ml)

Breakpoint on uconazole plates (g/ml)

CAGL-CDS (C. glabrata CDS)

Position (aa)a of Tn7 insertion

Protein length (aa)

Wild type Mutants ABC transporters Cgcdr1 Cgpdr16 Calcium transporters Cgcch1 Cgmid1 RNA polymerase II coactivators Cgspt20 Cgsrb8 Cgrgr1 Cgnut1 Cgrtg2 Nuclear ubiquitin complex Cgcdc34 Cgcdc53 ATPases Cgstv1 Cgvma13 Miscellaneous Cgynr047w Cgecm7 Cgpom152 Cginp53
a

16

16

4 8 8 8 8 8 8 8 16 8 8 8 4 8 8 8 8

4 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

CDS0106.1 CDS3202.1 CDS0026.1 CDS1553.1 CDS1138.1 CDS0156.1 CDS0391.1 CDS0322.1 CDS1501.1 CDS3640.1 CDS0757.1 CDS0587.1 CDS 2045.1 CDS0607.1 CDS2498.1 CDS0180.1 CDS0327.1

141 Promoter 1,137 316 22 745 67 502 280 281 360 213 89 107 183 114 182

1,499 344 2,124 591 685 1,358 1,037 1,099 600 295 822 910 505 895 437 1,317 1,096

aa, amino acids.

tant to 256 g of uconazole/ml (Table 2). One mutant for each locus (10 total) was chosen and analyzed as described in the following sections. We veried that the mutants we identied showed changes

in uconazole susceptibility as measured by the standard MIC assay (42). Overall, the breakpoint as measured in liquid correlated extremely well with that seen on plates (Tables 1 and 2). The one signicant exception was a mutant in the SDA1

TABLE 2. Mutants identied in a screen for uconazole resistance in C. glabrataa

Wild type or mutant Fluconazole MIC80 (g/ml) Breakpoint on uconazole plates (g/ml) CAGL-CDS (C. glabrata CDS) Position (aa) of Tn7 insertion Protein length (aa)

Wild type Mutants Ribosomal biogenesis Cgdbp3 Cgrpl16a Cgnop8 Mitochondrial biogenesis Cgsuv3 Cgmrpl4 Cgshe9 Others Cgsin3 Cgsda1 Cgpgs1 Cgycr051w
a

16

16

32 128 128 256 128 128 64 16 128 64

64 128 128 256 256 256 64 128 64 64

CDS1837.1 CDS4527.1 CDS1989.1 CDS1015.1 CDS3457.1 CDS2737.1 CDS0107.1 CDS0953.1 CDS2039.1 CDS4399.1

101 190 169 53 278 126 845 Promoter 45 84

540 190 514 724 315 405 1,493 745 506 206

aa, amino acids.

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FIG. 2. C. glabrata cch1 and mid1 mutants lose viability upon prolonged uconazole stress. (A) Growth of the C. glabrata cch1 and mid1 mutants is inhibited by uconazole. Wild-type cch1and mid1 mutant cells were grown at 30C in minimal medium supplemented with or without various amounts of uconazole (4 to 512 g/ml), and absorbance at 600 nm was monitored after 24 h of uconazole exposure. (B) Trypan blue exclusion assay for the viability assessment of the wild type and the cch1 and mid1 mutants in uconazole. Cells were grown at 30C in minimal medium supplemented with or without various amounts of uconazole as indicated. After 24 h of uconazole treatment, cells were stained with trypan blue for 5 to 10 min, and the number of stained (dead) and unstained (viable) cells was assessed microscopically. A minimum of 300 cells total (viable [unstained] and dead [stained] cells) were counted for each datum point. Cell survival data were plotted as the percentage of trypan blue exclusion and represent the mean of three experiments ( the standard deviation [SD]). (C) Viable cell count assay to assess the viability of wild type

gene that showed resistance on plates containing 128 g of uconazole/ml, whereas the breakpoint measured in liquid was no different than wild type (Table 2). Genes disrupted in mutants with altered uconazole susceptibility. In order to identify the genes disrupted in the various mutants, we rescued the Tn7 transposon and anking DNA for at least one mutant at each disrupted locus (see Materials and Methods). For each mutant, genomic DNA was isolated, digested, recircularized, and transformed into E. coli (8). The resulting circular plasmid contains the Tn7 transposon anked on either side by the gene it disrupts in the C. glabrata genome. Sequence analysis of the rescued plasmids with primers complementary to the ends of Tn7 revealed the identity of the disrupted genes. For the 17 susceptible and 10 resistant mutants, we regenerated the Tn7 insertion (see Materials and Methods) in the wild-type parental background. In all cases, the uconazole-susceptible or -resistant phenotype was regenerated, demonstrating that the Tn7 disruption was in fact responsible for the altered uconazole susceptibility phenotype (data not shown). The disrupted strains had no general growth defects. We measured the generation time for all 27 mutants in YPD media and found that, for 23 mutants, the range was 1.0 to 1.1 h, which was close to the wild-type generation time of 1.0 h. We found that nop8 (1.3 h), sin3 (1.2 h), sda1 (1.2 h), and vma13 (1.2 h) mutants had slightly longer doubling times (data not shown). The disrupted genes giving rise to sensitive phenotypes included genes encoding multidrug transporters, a calcium channel, components of RNA polymerase II mediator, and nuclear ubiquitin ligase complex (Table 1). The disrupted genes giving rise to resistant phenotypes included genes involved in phospholipid biosynthesis, transcription, and actin skeleton dynamics; three genes were involved in ribosomal biogenesis, and three were involved in mitochondrial assembly and function (Table 2). Disruption of CDR1 leads to a highly uconazole-susceptible phenotype. Fourteen mutants were susceptible to uconazole even at concentrations as low as 4 g/ml. One of these mutants was derived from a nonhomologous recombination event (as described in previous section) and was not analyzed further. The other 13 mutants were independent insertions derived from mutagenesis of a single fosmid F160; two of these were rescued. Both were Tn7 insertions in CDR1 (for Candida drug resistance 1) (57), a homologue of the S. cerevisiae multidrug transporter, PDR5 (2). The mutagenesis of fosmid F160 (carrying the CDR1 gene) had also resulted in one mutant showing a growth defect at 8 g of uconazole/ml (Fig. 1A)

and cch1 and mid1 mutants in uconazole. Cells were grown at 30C in minimal medium supplemented with or without various amounts of uconazole as indicated. After 24 h of uconazole treatment, the total number of cells was counted by using a hemocytometer, and the number of viable cells was determined by plating appropriate dilutions on YPD and YPG plates. CFU from YPG plates were counted as the nal readout, although essentially the same number of CFU was obtained on both YPG and YPD plates if small colonies (petites, as determined by their inability to grow on glycerol) on YPD plates were excluded from the nal viable cell counts. Cell survival data were plotted as the percentage of viability and represent the mean of three experiments ( the SD).

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and wild-type growth at 4 g of uconazole/ml. Sequence analysis revealed that this mutant contained a Tn7 insertion in the promoter region of the CDR1 gene, suggesting that the mild uconazole-sensitive phenotype of this mutant is due to a reduction in CDR1 transcription and thereby in the levels of Cdr1p. Mutants in the putative calcium channel (Cch1-Mid1p) show increased killing by uconazole. Two of our mutants contained insertions in the CCH1 and MID1 genes. These mutants were of particular interest to us since the S. cerevisiae homologues of CCH1 and MID1 are thought to encode subunits of a plasma membrane gated channel involved in Ca2 uptake (15, 23, 44). Cch1p is an N-glycosylated integral membrane protein which likely constitutes the catalytic subunit of the channel, whereas Mid1p is thought to be a regulatory subunit required for Cch1p function (36, 44). In C. albicans, functional calcineurin has been shown to be important for survival upon treatment with agents causing membrane stress, including azole agents (9, 60), and in S. cerevisiae, cch1 and mid1 mutants lose viability upon treatment with tunicamycin (which causes ER stress) (7). In light of these results, we examined whether the C. glabrata cch1 and mid1 mutants are unable to survive uconazole stress. We rst compared the growth of the cch1 and mid1 mutant and wild-type strains in liquid after 24 h in the presence of different concentrations of uconazole. At all concentrations of uconazole, all strainsthe wild type and the cch1 and mid1 mutantsshowed some growth (four to ve doublings) before uconazole inhibited the cell growth (data not shown) as measured by OD600. However, the nal cell density of the cch1 and mid1 mutant cells at all inhibitory concentrations of uconazole was consistently lower than that of wild-type cells, suggesting that cch1and mid1 cells either fail to replicate after a few generations or undergo cell death in the presence of uconazole. Consistent with their slightly increased susceptibility to uconazole, growth of the cch1 and mid1 mutants was signicantly inhibited at 8 g of uconazole/ml, whereas the growth of the wild-type strain remained unaffected at this concentration of uconazole (Fig. 2A). Next, we assessed the viability of wild-type, cch1, and mid1 cells after 24 h of growth in the presence of uconazole by a trypan blue exclusion assay. Cells were stained with trypan blue after uconazole exposure, and the percentage of staining cells was assessed microscopically. As shown in Fig. 2B, by this measure the cch1 and mid1 mutants displayed a signicantly lower viability than the wild-type strain after exposure to concentrations of uconazole ranging from 64 to 512 g/ml, suggesting that these mutants are killed at a higher rate than the wild type upon prolonged uconazole treatment. However, this difference was not seen at lower inhibitory concentrations of uconazole (8 or 16 g/ml) (Fig. 2B) nor when cells were exposed to uconazole (all concentrations) for short (8-h) time periods (data not shown). This observation suggests that in C. glabrata a functional Cch1-Mid1p channel is required for survival under long-term uconazole exposure. We made an independent more quantitative assessment of cell viability after uconazole treatment by quantitating the CFU of the cch1, mid1, and wild-type strains recovered in the presence of different concentrations of uconazole. C. glabrata wild-type, cch1, and mid1 cells were grown in minimal me-

FIG. 3. Trypan blue exclusion assay to assess the viability of all uconazole-susceptible mutants in uconazole. Cells were grown in minimal medium containing 512 g of uconazole/ml for 24 h and stained with trypan blue. A minimum of 300 cells total (viable [unstained] and dead [stained] cells) were counted microscopically for each mutant. Cell survival data were plotted as the percentage of trypan blue exclusion and represent the mean of two experiments ( the SD).

dium containing different concentrations of uconazole. After growth, total cell number was assessed by counting them with a hemocytometer, and the CFU were then measured by plating the cells on YPD or YPG plates (CFU were counted from YPG plates to exclude the petite [mitochondrion-incompetent] cells, since C. glabrata petites are uconazole resistant). No notable differences were seen between recoverable CFU of the cch1, mid1, or the wild-type strain when cells were grown at lower concentrations of uconazole (32 g/ml) (Fig. 2C and data not shown). However, consistently 10- to 20-fold lower CFU were observed for the cch1 and mid1 mutants compared to the wild-type strain when cells were treated with uconazole at concentrations from 64 to 512 g/ml (Fig. 2C), reconrming the earlier result (Fig. 2B) that both cch1 and mid1 cells are unable to survive the prolonged uconazole exposure. To classify our other uconazole-susceptible mutants, particularly in light of the relatively modest change in their uconazole susceptibility, we sought to determine whether any of the other uconazole-susceptible mutants also display a loss of viability upon uconazole treatment. We initially carried out a trypan blue exclusion assay on all mutants. C. glabrata cch1and mid1 mutants were used as positive controls in this assay. As shown in Fig. 3, most of the mutants (11 of 17 tested) displayed a wild-type-like survival rate of 90 to 95%. The cch1 and mid1 mutants exhibited 70 to 75% survival as determined by trypan blue exclusion assay, a nding consistent with earlier results, whereas four more mutantsecm7, ynr047w, pom152, and vma13were borderline cases, with 80 to 85% survival (Fig. 3). The results of the trypan blue assay were also conrmed by carrying out the viable count assay on two of the mutants that showed trypan blue exclusion of 80 to 85% (ecm7 and ynr047w) and two of the mutants that showed 90 to 95% trypan blue exclusion (cdc34 and pdr16). Viable counts from the cdc34, pdr16, ecm7, and ynr047w mutants ranged from 16 to 22%, which is essentially the same as for the wild-type strain (20% survival) (Fig. 2C and data not shown). Thus, a signicant loss of viability was seen only with the calcium channel mutant

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FIG. 4. Fluconazole and FK506 act synergistically and lead to cell death in wild-type C. glabrata cells. Cells were grown in minimal medium supplemented with either uconazole (64 g/ml) alone or uconazole (64 g/ml) plus FK506 (2 g/ml) as indicated. After 24 h of drug treatment, the total number of cells was counted by using a hemocytometer, and CFU counts were determined by plating cells onto YPG plates. Cell survival data were plotted as the percentage of viable cells in each culture and represent the mean of two experiments ( the SD).

strains. The loss of viability phenotype for the cch1 and mid1 mutants was not due to any enhanced uconazole susceptibility since all of the uconazole-susceptible mutants (including those with no loss of viability phenotype) showed similar susceptibility proles on plates and in liquid culture (Fig. 1, Table 1, and data not shown). Therefore, the loss of viability phenotype is a specic phenotype associated with a small subset of uconazole-susceptible mutants. Mutants in the putative calcium channel (Cch1-Mid1) are defective in Ca2 uptake upon uconazole exposure. Fluconazole and FK506 (a calcineurin inhibitor) in combination have been reported to act synergistically in a variety of yeast cells (9, 14, 37). In C. glabrata, a mild synergistic effect of the two drugs has been reported based on disk diffusion assays (9). Because of the role in S. cerevisiae of Cch1p and Mid1p in Ca2 uptake, we considered whether the loss of viability phenotype for the C. glabrata cch1 and mid1 mutants in our assays was also related to calcium signaling. We rst examined whether uconazole and FK506 act synergistically in C. glabrata. C. glabrata wild-type cells were grown in the presence of uconazole alone, FK506 alone, or uconazole and FK506 together, and the CFU were counted after 24 h of drug treatment. Consistent with the reports for other yeasts, a combination of uconazole and FK506 was found to be fungicidal since 10- to 15-foldfewer CFU were obtained when the cells were treated with both drugs versus when they were treated with uconazole alone (Fig. 4). No drop in viability was seen when cells were treated with FK506 alone (data not shown). This 10- to 15-fold drop in the viability of wild-type cells upon uconazole and FK506 treatment is of the same magnitude as the drop (relative to wild type) seen with cch1 and mid1 mutants treated with uconazole (Fig. 2C), a nding consistent with the mutants affecting the same pathway. In addition, the cch1 and mid1 mutants were no more susceptible than the wild-type strain to

treatment with a combination of uconazole and FK506 (Fig. 4), a result consistent with the cch1 and mid1 mutants being compromised in the calcineurin signaling pathway, targeted by FK506. To directly test whether the cch1 and mid1 mutants had defects in the transport of Ca2, we measured the accumulation of Ca2 in wild-type and mutant strains. ER stress has been shown to lead to increased Ca2 uptake in S. cerevisiae (7). First, we tested whether uconazole exposure would lead to the uptake of extracellular Ca2 in C. glabrata. C. glabrata wild-type cells were grown in the presence of uconazole, uconazole plus FK506, FK506 alone, or solvent (DMSO) alone. FK506 was used (to block calcineurin) because, in S. cerevisiae, calcineurin signals a feedback inhibition of the Cch1-Mid1p channel (36, 41). Total intracellular levels of Ca2 were measured after 1 to 4 h of drug treatment. Although no Ca2 accumulation was seen when cells were treated with FK506, a 10- to 12-fold increase in Ca2 uptake was seen for all strains when cells were grown in the presence of uconazole (Fig. 5). This Ca2 inux was signicantly elevated (70- to 75-fold) when wild-type cells were treated with both uconazole and FK506. However, no such increase in Ca2 levels was seen for the cch1 and mid1 mutants under similar conditions (Fig. 5), suggesting that these mutants are profoundly compromised for Ca2 uptake in response to azole stress. We also analyzed two other uconazole-susceptible mutants, rtg2 and spt20 (which do not exhibit loss of viability upon uconazole treatment), as negative controls. Both mutants exhibited the wild-type 60- to 90-fold increase in Ca2 uptake when treated with uconazole and FK506 together (data not shown). Fluconazole-resistant mutants affecting mitochondrial function. The C. glabrata mutants exhibiting the highest level of uconazole resistance (256 g/ml) had insertions in genes (SUV3, MRPL4, and SHE9) whose orthologues in S. cerevisiae

FIG. 5. Fluconazole exposure in C. glabrata induces Ca2 inux via Cch1-Mid1 channel. 45Ca2 accumulation in the wild type and the cch1 and mid1 mutants was measured after 4 h of treatment with either uconazole (64 g/ml) alone, FK506 (2 g/ml) alone, uconazole (64 g/ml) plus FK506 (2 g/ml), or DMSO (solvent) in YPD medium containing tracer amounts of 45CaCl2. The bars represent the mean of two experiments ( the SD); each experiment was performed in triplicate; thus, the bars represent the mean of six assays.

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FIG. 6. Fluconazole resistance of C. glabrata suv3, mrpl4, and she9 mutants is due to the loss of mitochondrial function. Various mutant strains and their uconazole-resistant derivatives (uR) were streaked on YPD and YPG plates, and growth was recorded after 2 days of incubation at 30C. Fluconazole-resistant derivatives of each mutant refer to the mutant colonies selected on YPD plates supplemented with uconazole.

are involved in mitochondrial function (Table 1) (12, 17, 38, 64). The corresponding S. cerevisiae mutants are all petites (17, 38, 64). We tested whether the C. glabrata counterparts (suv3, mrpl4, and she9, respectively) were also in fact petites. Surprisingly, only one of the C. glabrata mutants, suv3, was unable to grow on YPG plates, whereas mrpl4 and she9 were able to grow on YPG plates (Fig. 6). In conjunction with YPG growth assays, the function of mitochondria for the various mutants was assessed by staining cells with a mitochondria specic dye, Mito-Tracker Green FM-250. This dye is specically sequestered inside active mitochondria. Consistent with the growth data on YPG plates, bright staining of mitochondria was seen for C. glabrata wildtype cells and for the mrpl4 and she9 mutants, whereas the suv3 mutant showed only faint background staining, indicating the lack of functional mitochondria (data not shown). Although the C. glabrata mrpl4, she9, and suv3 mutants all showed the same apparent high level of uconazole resistance, only the suv3 mutant appeared phenotypically to be petite (Fig. 6). However, uconazole-resistant colonies arising from the C. glabrata mrpl4 and she9 did fail to grow on YPG plates (Fig. 6) showing that uconazole-resistant derivatives of these mutants were functionally petite. Thus, despite initial appearances, the uconazole resistance phenotype of the mrpl4 and she9 mutants is due to a loss of mitochondrial function. In contrast, the other seven uconazole-resistant mutants (both the parental mutant strains and the single colonies taken from uconazolecontaining plates for these same mutants) exhibited normal growth on YPG plates, indicating that they all had functional mitochondria (data not shown). Thus, the uconazole resistance phenotype for these other mutants is not due to the loss of mitochondrial function. From the data above, the apparent uconazole-resistant phenotype of the mrpl4 and she9 mutants was in fact due to a high frequency loss of mitochondrial function in these mutants. To quantify this, we carried out plating assays onto plates containing inhibitory concentrations of uconazole (64 to 512 g of uconazole/ml). As expected, the plating efciency of wild-type C. glabrata was low (ranging between 0.25 and 0.45%) at all inhibitory concentrations of uconazole. These

low-frequency uconazole-resistant colonies were functionally petite and unable to grow on YPG plates. This result is consistent with the ndings of Sanglard et al. (56), who originally reported that acquired uconazole resistance in C. glabrata is due to the loss of functional mitochondria (56). For the mrpl4 and she9 mutants, the plating efciency onto plates containing uconazole was approximately 10% (data not shown). For both mutant strains, the uconazole-resistant colonies were, as expected, functionally petite and unable to grow on YPG plates. Thus, our data suggested that the mrpl4 and she9 mutants, which display apparent very high levels of resistance to uconazole, are not in fact innately resistant to the drug but become resistant after losing mitochondrial function at a high frequency. Mechanisms of loss of mitochondrial function in wild-type and mutant C. glabrata. To gain an insight into the molecular mechanisms underlying the loss of mitochondrial competence in wild-type and mutant C. glabrata strains, we examined whether the petite phenotype of these derivative strains was reversible. We grew uconazole-resistant derivatives of the wild-type parent and of the mrpl4, she9, and suv3 mutant strains in YPD, plated the cells on YPG plates, and scored the number of colonies that are able to grow on YPG. We used a rho0 derivative of the wild-type strain as a control. This rho0 derivative was generated by treatment of the wild-type strain with ethidium bromide. Petite mutants generated in this fashion lack mitochondrial DNA. We found that the wt-rho0 strain, the suv3 mutant and its uconazole-resistant derivatives, and the uconazole-resistant derivatives of the she9 mutant were nonrevertible petites (data not shown). Seven of thirteen uconazole-resistant derivatives of the wild-type strain and three of six uconazole-resistant derivatives of the mrpl4 mutant were also nonrevertible petites. However, six of thirteen wildtype derivatives and three of six mrpl4 derivatives were able to revert to growth on YPG plates at a frequencies of 1.5 102 and 2.5 104, respectively (data not shown). For both strains, the mitochondrion-competent revertants were indistinguishable from the original nonpetite parental strains on the basis of their uconazole susceptibility proles (Fig. 7A).

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FIG. 7. Mitochondrion-associated acquired uconazole resistance in C. glabrata is reversible and is not associated with loss of the mitochondrial genome. (A) Revertants of uconazole-resistant derivatives of the C. glabrata wild type and mrpl4 mutant exhibit uconazole susceptibility like that of the original parental strain. Cells were grown in YPD medium overnight at 30C, and 5 l of 10-fold serial dilutions were spotted onto YPG and YPD plates with or without uconazole at a given concentration (64 and 256 g/ml) as indicated. Three independent revertant colonies (R1 to R3) were tested for each strain. Plates were photographed after 2 days of incubation at 30C. (B) The revertible uconazole-resistant derivatives of C. glabrata wild type and mrpl4 mutant possess mitochondrial genome. Nuclear and mitochondrial genomes were stained with DAPI as described in Materials and Methods, and cells were visualized by uorescence microscopy. Subpanel groups: I, reversible (wt-uR) and nonreversible (wt-rho0) uconazole-resistant derivatives of wild type; II, mrpl4 mutant and reversible (mrpl4-uR) uconazole-resistant derivative of mrpl4; III, she9 mutant and its nonreversible uconazole-resistant derivative she9-uR; IV, suv9 mutant, along with its nonreversible uconazole-resistant derivative (suv3-uR).

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Revertible uconazole resistance is not due to mitochondrial genome loss. We examined the various wild-type and mutant uconazole-resistant strains for the presence of the mitochondrial genome by DAPI staining. DAPI staining of the wild-type strain exhibited the wild-type pattern of a large nuclear genome and small dotted mitochondrial nucleoids (Fig. 7B). The ethidium bromide-derived wt-rho0 strain, which is a nonreverting petite, showed no staining of punctuate cytoplasmic bodies, indicating a loss of the mitochondrial genome (Fig. 7B). The suv3 petite strains are nonrevertible because the Suv3p protein product, disrupted by Tn7, is required for mitochondrial function and, as expected, these mutants still showed staining of mitochondrial nucleoids. Although the she9 mutant displayed distinct mitochondrial nucleoids upon DAPI staining, the derivative nonreverting uconazole-resistant colonies (she9-uR) lacked these staining nucleoids (Fig. 7C), indicating loss of the mitochondrial genome. That was also true for the nonreverting uconazole resistant derivatives of the wild-type and mrpl4 strain (data not shown). In contrast, the revertible uconazole-resistant derivatives of the wild-type strain (wt-uR) and of the mrpl4 mutant (mrpl4-uR) showed the normal DAPI staining pattern, with a large nuclear genome and small dotted mitochondrial nucleoids (Fig. 7B), demonstrating that the petite phenotype in these isolates is not due to the loss of mitochondrial genome loss. In conclusion, we showed that uconazole-associated loss of mitochondrial function, leading to acquired uconazole resistance, in wild-type C. glabrata is often reversible and, in these instances, not associated with the loss of the mitochondrial genome. This nding was reinforced by the behavior of the mrpl4 mutant, in which the fraction of cells able to become petite without losing the mitochondrial genome is much increased. DISCUSSION The innate uconazole resistance and the acquisition of elevated resistance in C. glabrata complicates the use of azole therapy for this organism. We have undertaken a functional genomic analysis addressing uconazole susceptibility in C. glabrata. By screening a library of 9,216 random insertion mutants (representing approximately 25% of C. glabarata genome) individually for their susceptibility to uconazole, we identied 27 genes that when mutated by Tn7 insertion alter susceptibility to uconazole. Mutations in three of these genes (CDR1, PDR16, and RTG2) have been previously demonstrated to result in changes in drug or azole susceptibility in various yeast species. The remaining 24 genes are novel and are implicated for the rst time in the azole susceptibility in any yeast. We tested the uconazole susceptibility of S. cerevisiae strains with deletions of the orthologues of the genes shown in Table 2 and found that of seven mutants tested (NOP8, PGS1, and SDA1 are essential genes in S. cerevisiae), only mutants in SUV3 and MRPL4 showed a slight (2-fold) increase in drug resistance (data not shown). This nding emphasizes the importance of carrying out analysis of drug resistance mechanisms in clinically relevant species. Azole-susceptible mutants. Of the 17 mutants that increase susceptibility to uconazole, two disrupt the multidrug efux pumps, Cdr1p and Pdr16p. Cdr1p belongs to the family of

ABC transporters and is a close homologue of S. cerevisiae Pdr5p and C. albicans Cdr1p (2, 4, 51). Cdr1p in C. glabrata has been implicated in uconazole resistance in vitro, and its expression has also been found to be signicantly elevated in many of clinical uconazole-resistant strains (57). PDR16 is another member of the ABC transporter family and is under the regulation by the pleiotropic drug resistance transcriptional factor, PDR1 (13, 68). In S. cerevisiae, PDR16 has been shown to regulate sterol metabolism and is specically required for resistance to the azole compounds miconazole and ketoconazole (68). Mutants of Cch1p and Mid1p, subunits of the putative plasma membrane channel, constitute the second interesting class of uconazole-susceptible mutants. Although mutants in these two genes were mildly susceptible to uconazole, they were notably defective in survival of long-term uconazole exposure, as measured by trypan blue exclusion and viable counts. Both the trypan blue exclusion assay and the viable count assay make clear that there is a signicant difference in survival of the wild-type and mutant strains; however, for the cch1 mutant, mid1 mutant, and wild-type strains, noteworthy differences lie in the numbers of calculated viable cells (recovered CFU) relative to cells that exclude trypan blue. For example, whereas the trypan blue assay shows that 90 to 95% of wild-type cells exclude dye after 24 h of uconazole treatment, only 15 to 20% of the cells are viable by the CFU assay (Fig. 2). This discrepancy likely means that the trypan blue assay underestimates the number of dead cells; alternatively, there may be signicant numbers of cells that are metabolically viable after uconazole treatment but which are unable to divide and form colonies when placed in nonselective media. In S. cerevisiae, Cch1p and Mid1p constitute the catalytic and the regulatory subunits of a high-afnity plasma membrane-based calcium channel (15, 23, 36, 44). Intracellular levels of Ca2 are important for many signaling cascades in both lower and higher eukaryotes. It has been postulated in S. cerevisiae, that membrane perturbation along with other stresses, leads to the inux of Ca2 either from the extracellular media or via mobilization of intraorganellar stores of Ca2 (14). In addition, the Ca2-dependent protein phosphatase, calcineurin, has been shown to be important for long-term survival under ER stress in S. cerevisiae and for the survival of membrane stress, virulence, and antifungal tolerance in C. albicans (5, 7, 9, 60). Although calcineurin, Cch1p, and Mid1p have been shown to be required for long-term survival of cells treated with agents causing ER stress in S. cerevisiae (7), this is the rst study reporting that uconazole treatment in C. glabrata leads to Cch1-Mid1-dependent Ca2 uptake and that this uptake is essential for survival upon prolonged uconazole treatment. Our ndings are consistent with those of Bonilla et al. (7), who reported that in S. cerevisiae Cch1-Mid1p was required for survival of ER stress and that FK506 treatment of C. glabrata resulted in sensitivity to tunicamycin. It is notable that the cch1 and mid1 mutants were not wholly unable to take up Ca2 in response to uconazole exposure (Fig. 5). Indeed, when exposed to uconazole alone, there was a marked increase in Ca2 uptake in wild-type and both mutant strains. In wild-type cells, there was an additional stimulation of Ca2 uptake in response to uconazole in the presence of FK506, which inhibits calcineurin. This additional

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stimulation depended entirely on the Cch1p-Mid1p channel since the mutant strains showed no such increase. Studies in S. cerevisiae have shown that the Cch1p-Mid1p channel is subject to a rapid downregulation of channel function in a calcineurindependent fashion. In S. cerevisiae, as in C. glabrata, Ca2 inux as a result of membrane stress can only be observed experimentally if feedback regulation of the channel is inhibited by FK506 (36, 41). Experimental considerations aside, our data shows that in C. glabrata, as in S. cerevisiae, there are CCH1/MID1-dependent and -independent mechanisms of Ca2 inux in response to uconazole. Based on the loss of viability seen in the cch1 and mid1 mutants, the Cch1-Mid1pdependent pathway is essential for azole stress survival. Lastly, ve of the uconazole susceptible mutants encode components of RNA polymerase II coactivator complexes. Srb8, Nut1, and Rgr1 are all components of Mediator (19, 28, 32, 35, 53). This complex of at least 20 proteins is required for activated transcription mediated by RNA polymerase II (28). This suggests that Mediator is required for the regulation of at least some of the genes important in the uconazole response. Potential targets of mediator include the ERG genes, as well as genes encoding drug transporters (CDR1 and PDH1/CDR2 in particular). Since the RNA polymerase II mediator mutants show only mildly uconazole-sensitive phenotypes, mediator is unlikely to be absolutely required for transcription of those genes. We also identied mutations in SPT20 and RTG2. Spt20 is a component of the SAGA complex which, among other activities, has histone acetyltransferase activity. Rtg2 is a cytosolic protein which in S. cerevisiae is involved in retrograde signaling from the mitochondria to the nucleus (33, 45). Recently, Rtg2p has been shown to be a constituent of the SLIK complex (SAGA-like complex), which is thought to be involved in chromatin modication and reorganization and is important in transcription by RNA polymerase II of certain loci (52). Intriguingly, rtg2 mutants in S. cerevisiae exhibit a mild sensitivity to cycloheximide and have a defect in the activation of multidrug resistance pumps in response to dysfunctional mitochondria (21). The identication of a rtg2 mutant with a mildly uconazole-sensitive phenotype suggests that a complex analogous to SLIK may be important in drug-induced activation of multidrug resistance pumps in C. glabrata. Mitochondrial dysfunction and acquisition of uconazole resistance. Three mutants, which exhibited the highest levels of uconazole resistance, were disrupted for mitochondrial function. Suv3p is a mitochondrial RNA helicase and is required for processing of mitochondrial RNA transcripts (64); suv3 mutant strains therefore inherently lack functional mitochondria. In the case of the mrpl4 and she9 mutants, the mutants had functional mitochondria but lost mitochondrial function efciently upon exposure to uconazole. She9p is an integral mitochondrial inner membrane protein and is required for the organization and biogenesis of mitochondria (38). She9p is also required for mitochondrial genome stability since she9 mutants lose their mitochondrial genome at an elevated frequency relative to the wild type. Mrpl4p is a putative structural component of mitochondrial large ribosomal subunit (17). Its disruption caused an increase in the rate of mitochondrial function loss but did not cause a notable destabilization of the mitochondrial genome. Our studies point out that mutants that do not themselves result in a petite phenotype can none-

theless be linked to acquired uconazole resistance if they result in an increased loss of mitochondrial function. We also found that loss of mitochondrial function occurs at an appreciable rate in wild-type cells. Dysfunctional mitochondria have been previously linked with drug resistance in both S. cerevisiae and C. glabrata (21, 56). We have extended these earlier studies by demonstrating that acquired uconazole resistance is not always due to the irreversible loss of mitochondrial genome but rather to a reversible loss of mitochondrial function. Sanglard et al. (56) analyzed upregulation of the multidrug transporter genes CDR1 and CDR2 in cultures of uconazole-associated petites. These authors reported, upon culturing of these isolates and concomitant with an increase in the growth rate, a downregulation of the transporter genes (56). This might be due to the reversion of their original respiratorydecient mutants (elevated levels of CDR1 and CDR2) to respiratory-competent cells (reduced CDR1 and CDR2 levels). Fluconazole-associated petites from the wild-type background can revert to respiratory competence at a rate of 2.5 102 in our study. This makes it highly unlikely that a uconazole-associated loss of mitochondrial competence is due to any genetic change and may suggest an epigenetic mechanism for switching between respiratory-competent and -incompetent states. In yeast, there are well established pathways of cross talk between dysfunctional mitochondria and the nucleus (33, 45). From a clinical perspective, there is a poor correlation in C. glabrata between the level of azole resistance measured in vitro and treatment success or failure. Epigenetic switching between mitochondrially competent (uconazole-susceptible) and incompetent (uconazole-resistant) states during infection might help explain how apparently sensitive isolates could be refractory to therapeutic treatment.
ACKNOWLEDGMENTS We thank Kyle Cunningham and members of the Cormack laboratory for helpful discussions during the course of this study. We are grateful to Jun Liu for the gift of FK506. This study was funded by the National Institutes of Health grant RO1 AI46223 to B.P.C.
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