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, 45 (4), 1174-83

Role of ATP-binding-cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida Glabrata

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Role of ATP-binding-cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida Glabrata

D Sanglard et al. Antimicrob Agents Chemother.

Abstract

Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753-2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 microg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 x 10(-4) to 4 x 10(-4)). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 microg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 microg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.

Figures

FIG. 1
FIG. 1
HFAR in C. glabrata strains DSY562, DSY1041, and DSY1613 following growth on YEPD containing fluconazole. The inoculum size of DSY562 on the YEPD plate containing fluconazole at 50 μg/ml was 3.3 × 104 cells. The frequency of HFAR cells in three independent experiments was 3.2 × 10−4 ± 0.71 × 10−4. Inoculum sizes for DSY1041 and DSY1613 were 4 × 104 and 5 × 104 cells, respectively. The frequency of HFAR cells from DSY1041 was similar to that obtained with DSY562. Incubation of the plates was for 4 days at 30°C. The fluconazole concentration used in YEPD is indicated for each plate.
FIG. 2
FIG. 2
Expression of ABC transporter genes in fluconazole-resistant isolates of C. glabrata. RNA was extracted from C. glabrata clinical isolates DSY562 and DSY565, from CgCDR1 deletion mutant DSY1041, and from HFAR cells obtained with DSY562 and DSY1041. The Northern blot was probed sequentially with 32P-labeled probes specific for all of the genes (CgCDR1, CgCDR2, CgSNQ2, and CgURA3) as indicated. The mRNA-hybridizing band detected in RNA of DSY1041-HFAR is probably due to cross-hybridization of the CgCDR1 probe with CgCDR2 mRNA, as discussed by Sanglard et al. (26). Due to variations in CgURA3-specific signals, EtBr-stained 28S RNA is shown to indicate that similar RNA quantities were loaded on the agarose gel. Using CgURA3 signals for normalization, CgCDR1 signals were increased 25- and 9-fold in DSY562-HFAR and DSY565, respectively, compared to those detected in parent strain DSY562.
FIG. 3
FIG. 3
Expression of multidrug efflux transporters in C. glabrata isolates with in vitro-acquired azole resistance. (A) Northern blot analysis. Approximately 5 μg of total RNA from each indicated yeast strain was loaded on the agarose gel. The Northern blot was hybridized sequentially with each 32P-labeled probe. The CgCDR2-specific signal from DSY1613 has a larger size than that from DSY1041 and probably corresponds to an aberrant RNA product. The Northern blots were revealed by exposure of Fuji XAR film at −80°C. Normalized signals for CgCDR1, compared to those detected in DSY562, were increased 7-, 62-, 67-, and 90-fold in DSY565, DSY562-rho, DSY565-rho, and DSY562-HFAR, respectively. Normalized signals for CgCDR2, compared to those detected in DSY562, were increased 1.7-, 1-, 68-, 40-, 22-, 26-, and 80-fold in DSY565, DSY1041, DSY562-rho, DSY565-rho, DSY1041-rho, DSY1041-HFAR, and DSY562-HFAR, respectively. (B) Immunodetection of CgCdr1p and CgCdr2p in cellular extracts. A 10-μg sample of total protein was loaded on a sodium dodecyl sulfate–10% (wt/vol) polyacrylamide gel and separated by electrophoresis. Western blots were incubated separately with CgCdr1p and CgCdr2p antisera, and signals were revealed by chemiluminescence on Fuji XAR film. Absence of signals in protein extracts from DSY1041 and DSY1613 is consistent with the deletion of CgCDR1 and CgCDR2 in these strains.
FIG. 4
FIG. 4
Susceptibility of C. glabrata multidrug efflux transporter mutants to azole derivatives. Each yeast strain is indicated at the left. Azole derivatives were added to YEPD medium at the indicated concentrations. Plates were incubated for 48 h at 30°C.
FIG. 5
FIG. 5
CgCDR1 and CgCDR2 expression in S. cerevisiae. Susceptibility tests of yeast transformants were performed with fluconazole and cycloheximide at the indicated concentrations. Plates were incubated for 48 h at 30°C.
FIG. 6
FIG. 6
Maintenance of azole resistance of HFAR cells in drug-free medium. (A) Expression of CgCDR1 and CgCDR2 in DSY562-HFAR in drug-free YEPD medium over subcultures A (no. 1) to P (no. 16). Intensities of CgCDR1 and CgCDR2 signals were normalized to those obtained with the CgURA3 probe. Normalized signals for CgCDR1, compared to those detected in DSY562, were increased 60-, 40-, 31-, 35-, 26-, 23-, and 8-fold in DSY562-A to -P, respectively, and 12-fold in DSY565. Normalized signals for CgCDR2, compared to those detected in DSY562, were increased 90-, 33-, 22-, 22-, 18-, 15-, and 5-fold fold in DSY562-A to -P, respectively, and 2.5-fold in DSY565. (B) Growth of clinical isolate DSY562, DSY565 (azole-resistant), and DSY562-HFAR subcultures (A to P) in YEPD.
FIG. 7
FIG. 7
Staining of mitochondria in C. glabrata. Strains DSY562, DSY565, DSY1041, and DSY1613 are respiratorily competent and therefore were designated rho+. From each of these strains, HFAR cells obtained by fluconazole exposure or rho0 cells obtained by EtBr treatment were incubated with MitoTracker Green FM and examined by either phase-contrast (top rows) or epifluorescence (bottom rows) microscopy.
FIG. 8
FIG. 8
Mitochondrial loss is linked to the acquisition of azole resistance in C. glabrata. Wild-type, HFAR, and rho0 cells were spotted in serial dilutions onto YEPD agar and fluconazole-containing YEPD medium. Plates were incubated for 48 h at 30°C.

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