JAC Advance Access published online on October 2, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm321
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The Candida glabrata putative sterol transporter gene CgAUS1 protects cells against azoles in the presence of serum
1 Department of Chemistry and Biochemistry, Suzuka National College of Technology, Shiroko, Suzuka, Mie 510-0294, Japan 2 Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 3 Department of Biology, Indiana University-Purdue University Indianapolis, 723 W. Michigan St., Indianapolis, IN 46202, USA 4 Department of Electronic and Information Engineering, Suzuka National College of Technology, Shiroko, Suzuka, Mie 510-0294, Japan 5 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
* Corresponding author. Tel: +81-59-368-1835; Fax: +81-59-368-1820; E-mail: nakayama{at}chem.suzuka-ct.ac.jp
Received 9 March 2007; returned 21 April 2007; revised 31 July 2007; accepted 1 August 2007
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Objectives: The uptake of endogenous sterol from serum may allow Candida glabrata to survive azole treatment. This study aims to determine the contribution of a sterol transporter that alters fluconazole sensitivity in the presence of serum.
Methods: Bioinformatic analysis predicted CgAUS1 as the C. glabrata orthologue of the Saccharomyces cerevisiae transporters AUS1 and PDR11. To investigate whether the CgAUS1 gene has sterol transporter activity, we investigated the effects of an AUS1 deletion on the growth of a tetracycline-regulatable ERG9 strain (tet-ERG9aus1), wherein ERG9 expression is turned off giving rise to a sterol requirement. Tetracycline-dependent repression of CgAUS1 in the tet-AUS1 strain was used to determine the fluconazole susceptibility of CgAUS1 in the presence and absence of serum.
Results: The tetracycline-treated tet-ERG9aus1 strain failed to grow in the presence of serum, whereas the parental tet-ERG9AUS1 strain grew by incorporating sterol from exogenously supplied serum. Serum cholesterol protected cells against the antifungal effects of fluconazole and this protection was lost by repressing CgAUS1 gene expression. Furthermore, such protection was also observed during itraconazole treatment, but not observed in cells treated with non-azole antifungals.
Conclusions: CgAUS1 appears to function as a sterol transporter that may contribute to lower azole susceptibility in the presence of serum and to protect C. glabrata against azole toxicity in vivo.
Key Words: antifungal resistance , sterol uptake , tetracycline-regulatable strain
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Introduction |
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Azole antifungals are fungistatic inhibitors of ergosterol biosynthesis and have been widely used for the treatment of both systemic and superficial fungal infections. Prolonged use can select for azole-resistant clinical isolates. It has become a concern that azole-resistant isolates are recovered from immunocompromised and immunosuppressed patients with increased frequency. Therefore, the mechanism of azole resistance in Candida albicans has been well studied; resistance may be due to overexpression of drug efflux pumps, overexpression or alteration in the azole target Erg11p or mutation of a gene such as ERG3 that participates later in the ergosterol biosynthesis pathway.1–5
The pathogenic fungus Candida glabrata is the second or third most common cause of candidosis. Infections by this organism have been linked to the death of immunocompromised and immunosuppressed patients.6,7 Unlike C. albicans, C. glabrata grows only as the yeast form in the host and lacks certain virulence attributes: secreted hydrolases are minimal and adhesion is relatively weak.8–13 A potential explanation of why C. glabrata infections are now prevalent is its intrinsically low susceptibility to azoles. In a four and one-half year global surveillance study, 
18.9% of 8013 C. glabrata clinical isolates were found to be resistant to fluconazole, whereas only 0.95% of 52 987 C. albicans isolates and 3.4% of 4582 Candida tropicalis isolates were found to be resistant.14
Saccharomyces cerevisiae cells, which are closely related to C. glabrata cells,15 do not take up exogenous sterols, a phenomenon called ‘aerobic sterol exclusion’. Under anaerobic conditions or in mutants that lack haem, this yeast requires sterols and unsaturated fatty acids, as the synthesis of these lipids requires molecular oxygen.16,17 Recent studies indicated that even under aerobic conditions, growth of C. glabrata cells depleted of cellular ergosterol was rescued by adding human or mouse serum to the medium.18 In addition, it has been reported that mutant clinical isolates unable to synthesize ergosterol could take up cholesterol from the growth medium.19,20 Thus, sterol uptake by C. glabrata clinical isolates might contribute to their resistance to azoles. In order to investigate this hypothesis, we searched for a predicted sterol transporter gene in C. glabrata and studied its contribution to azole resistance. Herein, we report that expression of a C. glabrata sterol transporter gene CgAUS1 could overcome growth inhibition by fluconazole in the presence of serum, suggesting a novel mechanism to reduce the susceptibility to azoles.
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Strains and media
Escherichia coli DH5
[F–, 
80, lacZ
M15, (lacZYA-argF) U169, hsdR17(rk– mk+], recA1, endA1, deoR, thi-1, supE44, gyrA96, relA1 
–) was used in plasmid propagation. Bacterial strains were grown in Luria-Bertani medium (LB) with ampicillin. The C. glabrata strains used in this study are listed in Table 1. ACG4 and ACG22 strains were used to generate conditional mutants.21 Strains were grown in Yeast-Peptone-Dextrose (YPD) medium [1% Bacto yeast extract (Difco), 2% Bacto peptone (Difco) and 2% glucose] or in Complete Supplement Mixture (CSM) medium (pH 5.8) [0.67% Bacto yeast nitrogen base without amino acids (Difco), 0.079% CSM (MP Biomedicals, Inc., OH, USA), 2% glucose and 40 mg/L additional adenine and 60 mg/L additional histidine]. Solid medium was supplemented with 2% agar (Nacalai Tesque, Kyoto, Japan). Doxycycline and bovine serum were purchased from Sigma-Aldrich Chemical Co. (St Louis, USA). Bile and cholesterol were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
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Plasmid and strain construction
Strain tet-ERG9AUS1, in which the endogenous ERG9 promoter in ACG2221 was replaced with the tetracycline-regulatable promoter 97t,21 was generated by using homologous recombination to sandwich the CgURA3-97t DNA fragment between the 5' non-coding region of the ERG9 gene (nt –503 to –133) and the 5'-portion of the ERG9 ORF (nt –6 to 314). The integration of the expected locus in transformants was confirmed by PCR using primers E9CHA (5'-GCTGTGATCCACGATGAGC-3') and E9CHB (5'-ACCTGCAAGGACAATCAATTC-3') (data not shown).
The Cgaus1 deletion strain, tet-ERG9aus1, was obtained by transforming tet-ERG9AUS1 with a fragment in which the CgHIS3 gene was flanked by the 5' non-coding (nt –479 to –46) and the 3' non-coding regions (nt +4194 to +4594) of the CgAUS1 gene. The integration of the HIS3 gene at the expected locus was confirmed in transformants by PCR using primers AUS1CHA (5'-CGTACAAGTGTCCTGCCATTCG-3') and CgAUS1TETCHB (5'-AATCCAAAGCGGTTGAGGAATCC-3') (data not shown).
The Cgaus1 conditional strain, tet-AUS1, was obtained by transforming ACG4 with a fragment in which the HIS3-97t gene was flanked by the 5' non-coding (nt –479 to –46) and the 5' coding sequence regions (nt –6 to +464) of the CgAUS1 gene. The integration of the HIS3-97t gene at the expected locus was confirmed in transformants by PCR using the primers AUS1CHA and AUS1CHB (5'-GCTTATACGAGGCGAACCTGAG-3') (data not shown).
Nucleotide/amino acid sequence analyses and homology searches were performed using the BLAST search, the CLUSTAL W(1.83) multiple/pairwise sequence alignment program and the T-Coffee (3.27) multiple/pairwise sequence alignment program.22 The synteny and transcriptional factors were analysed using our scripts implemented in Ruby (http://www.ruby-lang.org/) and BioRuby (http://bioruby.org/). The synteny around a targeted gene was analysed by comparing chromosomal genes near the target gene with adjacent genes close to homologues corresponding to the target gene. The C. glabrata AUS1 homologue was the closest match in a BLAST search. The nucleotide sequences were obtained from GenBank (http://www.ncbi.nih.gov/) for C. glabrata and S. cerevisiae.
Northern analysis for CgAUS1 transcript
Cells grown on YPD medium at 30°C overnight were inoculated at 1 x 106 cells/mL and cultured for 4 h in CSM (pH 5.8) medium with and without 10% bovine serum in the absence or presence of 20 mg/L doxycycline or 5 mg/L fluconazole. Total RNA samples were prepared as described previously.23 Total RNA was separated on a 0.9% formamide agarose gel and transferred to Hybond-N+ nylon membrane (GE Healthcare UK Ltd, Buckinghamshire, UK). Using ACG4 chromosomal DNA as template, DNA probes for detection of CgAUS1 and ACT1 were amplified with primers CgAUS1-5' (5'-GCTCAGAAGTTGATTTATACACCCA1-3') and CgAUS1-3' (5'-CCAATCAAGATACCAAAGTTTCTCCA-3'), CgACT1-5' (5'-GTGACGATGCTCCAAGAGCCGTCT-3') and CgACT1-3' (5'-CCACTTTCGTCGTATTCTTGCTTGGA-3'), respectively. Amplified DNAs were labelled with AlkPhos Direct Labelling Module (GE Healthcare UK Ltd). After hybridizing with either CgAUS1 or ACT1 probes for 18 h, signals were detected according to manufacturer's instructions with CDP-Star Detection Reagent (GE Healthcare UK Ltd).
The sterol content of C. glabrata strains was determined according to previously described procedures.24 Cells were grown on CSM with or without 10% bovine serum (53 mg/L total cholesterol after saponification). Sterols were analysed by gas chromatography. An HP5890 series II equipped with the Hewlett-Packard CHEMSTATION software package (CA, USA) was used to analyse sterol content. The capillary column (DB-5) was 15 m x 0.25 mm x 0.25 µm film thickness (J&W Scientific, CA, USA) and was programmed from 195 to 280°C (1 min at 195°C, then an increase to 240°C at 20°C/min, followed by 2°C/min until a final temperature of 280°C was reached). The linear velocity was 30 cm/s using nitrogen as the carrier gas, and all injections were run in the splitless mode. Sterol analysis showed that cholesterol was the only sterol in bovine serum used in this study.
Approximately 5 x 105 cells were used to inoculate CSM medium (2 mL) and were then cultured with or without 10% bovine serum in the presence or absence of doxycycline (100 mg/L). The culture OD660 was measured after 14 h at 30°C with Bio-Rad Laboratories SmartspecTM 3000 (Hercules, CA, USA). Samples (100 µL) of diluted cells were spread on YPD and cfu was determined after 24 h of incubation at 30°C. Microscopic observations suggested that growth defects were not due to clumping of the strains.
Approximately 2 x 103 cells were used to inoculate CSM medium (200 µL) and were then cultured with serially diluted fluconazole (from 0 to 400 mg/L; Pfizer), itraconazole (from 0 to 40 mg/L; Sigma-Aldrich Chemical Co.), terbinafine (from 0 to 40 mg/L; Novartis, Basel, Switzerland), micafungin (from 0 to 4 mg/L; Astellas Pharma Inc., Osaka, Japan) or amphotericin B (from 0 to 32 mg/L; Sigma-Aldrich Chemical Co.) in the presence or absence of doxycycline (20 mg/L) and 10% bovine serum. After incubation at 30°C for 24 or 48 h, the OD at 595 nm of the cells was measured to determine antifungal susceptibility with a Beckman Coulter DTX880 Multimode Detector (Fullerton, CA, USA). No growth of C. glabrata was observed in RPMI1640 serum-containing media (human, fetal bovine or bovine).
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Identification of a sterol transporter in C. glabrata
The sterol transporter genes, AUS1 and PDR11, which are expressed anaerobically in S. cerevisiae,25 were selected as queries for homology searches to identify sterol transporters in C. glabrata. A BLAST search against the C. glabrata genome26 (http://cbi.labri.fr/Genolevures/elt/, CAGL) predicted CAGL0F01419 g to be a functional homologue of both S. cerevisiae genes. As conserved gene locations on chromosomes correlate with evolutionary relationships, the homology of genes adjacent to CAGL0F01419g was compared with those adjacent to S. cerevisiae AUS1 or S. cerevisiae PDR11. As a result of this synteny analysis, some homologues adjacent to CgAUS1 on C. glabrata chromosome F were found to be located adjacent to S. cerevisiae AUS1 on chromosome XV in a conserved order. In contrast, no homologues adjacent to CgAUS1 were located adjacent to S. cerevisiae PDR11 on chromosome IX (Figure 1). In addition, the predicted amino acid identity of CAGL0F01419g with S. cerevisiae AUS1 (71.3%) was greater than that with PDR11 (64.4%) (Table 2). The predicted gene was therefore named CgAUS1 (C. glabrata ABC transporter involved in the uptake of sterol, accession no. AB 297452). The predicted ORF is 4197 bp with no intron and encodes a putative protein, CgAus1p, of 1398 amino acid residues (158 kDa). The sequence of CgAus1p suggests a typical fungal ABC transporter, arranged in two halves, each half comprising an N-hydrophilic domain (containing the ATP-binding cassette) and a C-hydrophobic domain with six transmembrane domains.27
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Effect of CgAUS1 gene expression on the growth of a sterol auxotrophic mutant
As described previously,18 repression of ERG9 expression using the tetracycline-regulated 97t21 promoter in the tet-ERG9AUS1 strain blocked squalene biosynthesis and growth.18 Inclusion of serum in the medium rescued the growth defect in a tet-off ERG9 strain (97SQS).18 To determine whether CgAUS1 encodes a putative sterol transporter, the Cgaus1 deletant strain, tet-ERG9aus1, was generated from tet-ERG9AUS1. When the ERG9 gene products of both tet-ERG9AUS1 and tet-ERG9aus1 were depleted by doxycycline, cells showed a severe growth defect in the absence of bovine serum because of the inability to synthesize squalene. In contrast to near-normal growth of the tet-ERG9AUS1 strain grown in the presence of doxycycline and bovine serum, tet-ERG9aus1 cells were not rescued under the same conditions, apart from a barely significant increase in cfu (Figure 2). These results are consistent with the sterol uptake in the CgAUS1 strain, compensating effectively for endogenous sterol depletion.
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Manipulation of sterol content in the tet-AUS1 strain
To confirm this finding, we generated the conditional mutant tet-AUS1 from ACG4,21 in which the CgAUS1 gene was placed under the control of a tetracycline-regulatable promoter.21 When tet-AUS1 cells were cultured with doxycycline, growth was unaffected both in the presence or in the absence of bovine serum, indicating that expression of the CgAUS1 gene did not affect growth under these conditions (data not shown). The CgAUS1 gene in tet-AUS1 was constitutively expressed in the absence of doxycycline whether the cells were cultured with or without bovine serum, but completely repressed in the presence of doxycycline (Figure 3). The effects of CgAUS1 expression on sterol uptake were determined by measuring the sterol composition of tet-AUS1 cells. When tet-AUS1 cells were cultured with bovine serum and without doxycycline, more than one half of the cellular sterol was cholesterol (Table 3). Cholesterol accumulation in tet-AUS1 cells grown with bovine serum decreased with increasing doxycycline concentrations. Furthermore, no cholesterol was detected in tet-AUS1 cells grown in medium not containing serum either with or without doxycycline (Table 3), supporting the hypothesis that the CgAus1p facilitates cholesterol uptake from serum.
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Effects of CgAUS1 gene knockdown on fluconazole susceptibility
Because cholesterol uptake from serum alleviated the growth defect of ERG11-depleted cells,28 we hypothesized that this uptake would reduce the susceptibility of C. glabrata to fluconazole, even though Erg11p was inhibited by the drug. Measurement of the azole susceptibility of the tet-AUS1 strain and its parent ACG4 provided a test for this hypothesis (Figure 4). The addition of bovine serum allowed growth to occur at fluconazole concentrations of >25 mg/L for both ACG4 and tet-AUS1 cells without doxycycline treatment. When tet-AUS1 was cultured with bovine serum, cells were more susceptible to fluconazole at >12.5 mg/L in the presence of doxycycline. In contrast, the growth inhibition profile of ACG4 was not altered by adding doxycycline in the presence of fluconazole (Figure 4a and b). Northern analysis revealed that 20 mg/L doxycycline also repressed CgAUS1 expression in tet-AUS1 cells and that the CgAUS1 gene was constitutively expressed in the presence of fluconazole. In contrast, CgAUS1 expression was up-regulated by adding bovine serum in strain ACG4 that has the native promoter of CgAUS1 (Figure 3).
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We further tested substitutes of bovine serum to rescue growth inhibition of tet-AUS1 by fluconazole. Although addition of free cholesterol (54 mg/L) did not alter the growth inhibition profile of fluconazole, the addition of 0.8% bile reduced fluconazole susceptibility of both ACG4 and tet-AUS1 to a greater degree than bovine serum (Figure 4c–f).
Effects of CgAUS1 gene knockdown on antifungal susceptibility
To confirm whether CgAUS1 expression could affect susceptibility to other azoles and antifungals, we tested whether depleting the CgAUS1 gene product would alter susceptibility to the other antifungals itraconazole (Figure 5a and b), amphotericin B (Figure 5c and d), terbinafine (Figure 5e and f) and micafungin (Figure 5g and h). The susceptibility of tet-AUS1 cells for all drugs was decreased when the cells were cultivated with bovine serum in the absence of doxycycline (Figure 5). The cells of tet-AUS1 cultured with doxycycline and bovine serum were only more susceptible to itraconazole. Thus, tet-AUS1 did not show increased susceptibility to non-azole compounds even when CgAUS1 expression was repressed (Figure 5), suggesting that serum uptake only alleviated azole sensitivity.
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Molecular genetic manipulation of the expression of the ERG9 and CgAUS1 genes demonstrates that the CgAUS1 gene product functions as a sterol transporter that ameliorates the growth inhibition caused by the loss of ERG9 expression or fluconazole-mediated inhibition of Erg11p. Three different experiments support these conclusions. First, the growth of doxycycline-treated tet-ERG9AUS1 cells was rescued in serum-supplemented medium when ERG9 was repressed, whereas serum did not rescue the severe growth defect of a doxycycline-treated tet-ERG9aus1 strain. Secondly, the tet-AUS1 did not accumulate cholesterol from serum-supplemented media under CgAUS1-repressed conditions. Thirdly, expression of CgAUS1 ameliorated the growth inhibitory effects of azole in the presence of serum.
Although the drug susceptibility tests demonstrated that CgAUS1 expression would affect azole susceptibility, two unexpected results were observed. One was residual growth of tet-AUS1 cultured with serum in the presence of doxycycline as growth inhibition did not fall to <20% (Figure 4). Another was trailing of the growth inhibition curve of tet-AUS1 cultured with serum in the presence of doxycycline at higher concentrations of itraconazole (Figure 5a and b). In this case, growth inhibition gradually diminished at concentrations >12.5 mg/L. Although further studies are required to explain such phenomena, some possibilities are as follows. Overexpression of efflux pumps such as CDR1 might be raised as an explanation for trailing, as previous studies have reported that higher concentration of azoles induces overexpression of efflux pumps such as CDR1.29 As for residual growth, two possibilities can be suggested: (i) constituents from serum that control a key function affecting azole susceptibility might be transported by some other transporters; and (ii) a small amount of sterol might support residual growth. As shown in Table 3, cholesterol was detected in tet-AUS1 cells cultured in the presence of doxycycline. These results suggest that sterol transport might not be completely inhibited as any gene knockdown system may not completely abolish target gene expression. An alternative reason for cholesterol detection might be that other transporters may recognize cholesterol as a lower affinity substrate [transporters such as SNQ2 (CAGL0I04862g) homologue are highly similar to CgAUS1].
The susceptibility testing of antifungals in serum-containing media demonstrated that CgAus1p did not affect susceptibility to non-azole antifungals such as amphotericin B, terbinafine and micafungin (Figure 5c–h). Our previous studies demonstrated that C. glabrata cells still continue to synthesize ergosterol even while taking up exogenous sterols.18,28 The effective concentration of terbinafine may have decreased because of binding to constituents of serum30 both in ACG4 and in tet-AUS1 cells (Figure 5e and f). Furthermore, terbinafine susceptibility was altered by adding 20 mg/L doxycycline both in ACG4 and in tet-AUS1 in the presence of bovine serum, suggesting that doxycycline may affect drug permeability. In fact, 100 mg/L doxycycline also lowered fluconazole susceptibility of ACG4 in the presence of serum, and CgAUS1 expression in ACG4 was repressed under these conditions (data not shown); however, 20 mg/L doxycycline did not affect ACG4 fluconazole susceptibility or CgAUS1 expression (Figure 3a and b).
Xiong et al.31 recently demonstrated that Aspergillus fumigatus also imports exogenous cholesterol from serum and that this property counteracted the toxicity of sterol biosynthetic inhibitors. Interestingly, it is noted that both C. glabrata and A. fumigatus can take up exogenous sterols under aerobic conditions, whereas S. cerevisiae, which is phylogenetically closely related to C. glabrata,15 takes up sterols under anaerobic conditions or in mutants lacking haem.15,16 As shown in Figure 3, the CgAUS1 gene was up-regulated by serum in strain ACG4 that has the native CgAUS1 promoter, suggesting that C. glabrata may have an enhanced growth response to constituents in serum. However, the aerobic uptake of cholesterol appears to require a serum/bile factor, as free cholesterol is not taken up by tet-AUS1 strain constitutively expressing CgAus1p (Figures 3 and 4). For example, lipoproteins may need to associate with the transporter for effective cholesterol transport,18–20,28,31 or alternatively, other uncharacterized factors may play a role similar to the cell wall protein Dan1p in S. cerevisiae that is specifically produced under anaerobic conditions and required for sterol uptake along with Aus1p and Pdr11p.32 Promoter analysis for AUS1, PDR11 and CgAUS1 also supports different gene regulation of sterol transporter genes in C. glabrata and S. cerevisiae. The Ecm22p promoter-binding motif, which is the major regulator of the S. cerevisiae AUS1 gene,33 is not present in the promoter region of CgAUS1, which suggests that CgAUS1 is not an anaerobically expressed gene in C. glabrata as in S. cerevisiae.
Our results, together with studies of some other pathogenic fungi, suggest that sterol transporters can counteract ergosterol deficiency and azole toxicity. It may also be possible that mutations in regulatory factors of various pathogenic fungi, whose sterol transporter(s) is repressed in vivo, might allow the development of azole resistance. Thus, the study of sterol transporters in pathogenic fungi, including their physiological functions, regulation and mutation, can provide insights into novel mechanisms of azole resistance and could promote the development of improved antifungals.
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This work was supported by grant-in-aid from SFIF (Systemic Fungus Infection Forum). K. T. is supported by grants KH33310 and SH24405 from the Japan Health Sciences Foundation. M. N. is supported by the Health Science Research Grants for Research on Emerging and Re-emerging Infectious Diseases of the Ministry of Health, Labour and Welfare of Japan. M. B. acknowledges the support of NIH grant GM62104 and Burroughs Wellcome Fund grant.
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We declare no conflicting financial interests.
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These authors have contributed equally. 
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Acknowledgements |
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We thank Drs Masayuki Sudoh and Kunio Kitada for providing strains ACG4 and ACG22, Astellas Pharma Inc., for providing micafungin, Mr Tetsuo Iwata, Takuya Nakanishi and Makoto Okano for experimental help and Professor Richard Cannon and Maureen Bard for critical reading of the manuscript.
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References |
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1 . Casalinuovo IA, Di Francesco P, Garaci E. Fluconazole resistance in Candida albicans: a review of mechanisms. Eur Rev Med Pharmacol Sci (2004) 8:69–77.[Medline]
2
.
Chau AS, Gurnani M, Hawkinson R, et al. Inactivation of sterol 5,6-desaturase attenuates virulence in Candida albicans. Antimicrob Agents Chemother (2005) 49:3646–51.
3
.
Ghannoum MA, Rice LB. Antifungal agents: mode of action, mechanism of resistance and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev (1999) 12:501–17.
4 . Vanden Bossche H, Dromer F, Improvisi I, et al. Antifungal drug resistance in pathogenic fungi. Med Mycol (1998) 36(Suppl 1):119–28.[CrossRef][Medline]
5
.
White TC, Marr KA, Bowden RA. Clinical, cellular and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev (1998) 11:382–402.
6 . Fortun J, Lopez-San Roman A, Velasco JJ, et al. Selection of Candida glabrata strains with reduced susceptibility to azoles in four liver transplants patients with invasive candidiasis. Eur J Clin Microbiol Infect Dis (1997) 16:314–8.[CrossRef][Web of Science][Medline]
7
.
Fidel PL, Vazquez JA Jr, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev (1999) 12:80–96.
8 . al-Rawi N, Kavanagh K. Characterization of yeasts implicated in vulvovaginal candidosis in Irish women. Br J Biomed Sci (1999) 56:99–104.[Web of Science][Medline]
9 . Al-Hedaithy SSA. Spectrum and proteinase production of yeasts causing vaginitis in Saudi Arabian women. Med Sci Monit (2002) 8:498–501.
10 . Kantarcioglu AS, Yucel A. Phospholipase and protease activities in clinical Candida isolates with reference to the sources of strains. Mycoses (2002) 45:160–5.[CrossRef][Web of Science][Medline]
11 . Krcmery V, Barnes AJ. Non-albicans Candida spp. causing fungemia: pathogenicity and antifungal resistance. J Hosp Infect (2002) 50:243–60.[CrossRef][Web of Science][Medline]
12 . Moran GP, Sullivan DJ, Coleman DC. Emergence of non-Candida albicans Candida species as pathogens. In: Candida and Candidiasis—Calderone RA, ed. (2002) Washington, DC: ASM Press. 37–53.
13 . Nikawa H, Egusa H, Makihira S, et al. A novel technique to evaluate the adhesion of Candida species to gingival epithelial cells. Mycoses (2003) 46:384–9.[CrossRef][Web of Science][Medline]
14
.
Hazen KC, Baron EJ, Colombo AL, et al. Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion. J Clin Microbiol (2003) 41:5623–32.
15
.
Barns SM, Lane DJ, Sogin ML, et al. Evolutionary relationships among pathogenic Candida species and relatives. J Bacteriol (1991) 173:2250–5.
16 . Andreasen AA, Stier TJ. Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Physiol (1953) 41:23–36.[CrossRef][Web of Science][Medline]
17
.
Lewis TA, Taylor FR, Parks LW. Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae. J Bacteriol (1985) 163:199–207.
18
.
Nakayama H, Izuta M, Nakayama N, et al. Depletion of the squalene synthase (ERG9) gene does not impair growth of Candida glabrata in mice. Antimicrob Agents Chemother (2000) 44:2411–8.
19 . Bard M, Sturm AM, Pierson CA, et al. Sterol uptake in Candida glabrata: rescue of sterol auxotrophic strains. Diagn Microbiol Infect Dis (2005) 52:285–93.[CrossRef][Web of Science][Medline]
20 . Hazen KC, Stei J, Darracott C, et al. Isolation of cholesterol-dependent Candida glabrata from clinical specimens. Diagn Microbiol Infect Dis (2005) 52:35–7.[CrossRef][Web of Science][Medline]
21
.
Nakayama H, Izuta M, Nagahashi S, et al. A controllable gene-expression system for the pathogenic fungus Candida glabrata. Microbiology (1998) 144:2407–15.
22 . Notredame C, Higgins DG, Heringa J. T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol (2000) 302:205–17.[CrossRef][Web of Science][Medline]
23
.
Hanaoka N, Umeyama T, Ueno K, et al. A putative dual-specific protein phosphatase encoded by YVH1 controls growth, filamentation and virulence in Candida albicans. Microbiology (2005) 151:2223–32.
24
.
Gachotte D, Sen SE, Eckstein J, et al. Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation. Proc Natl Acad Sci USA (1999) 96:12655–60.
25
.
Wilcox LJ, Balderes DA, Wharton B, et al. Transcriptional profiling identifies two members of the ATP-binding cassette transporter superfamily required for sterol uptake in yeast. J Biol Chem (2002) 277:32466–72.
26 . Dujon B, Sherman D, Fischer G, et al. Genome evolution in yeasts. Nature (2004) 430:35–44.[CrossRef][Medline]
27 . Jungwirth H, Kuchler K. Yeast ABC transporters—a tale of sex, stress, drugs and aging. FEBS Lett (2006) 580:1131–8.[CrossRef][Web of Science][Medline]
28
.
Nakayama H, Nakayama N, Arisawa M, et al. In vitro and in vivo effects of 14-demethylase (ERG11) depletion in Candida glabrata. Antimicrob Agents Chemother (2001) 45:3037–45.
29 . Niimi M, Nagai Y, Niimi K, et al. Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole. J Chromatogr B Analyt Technol Biomed Life Sci (2002) 782:245–52.[CrossRef][Web of Science][Medline]
30 . Ryder NS, Frank I. Interaction of terbinafine with human serum and serum proteins. J Med Vet Mycol (1992) 30:451–60.[Web of Science][Medline]
31
.
Xiong Q, Hassan SA, Wilson WK, et al. Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors. Antimicrob Agents Chemother (2005) 49:518–24.
32
.
Li Y, Prinz AW. ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulated sterol movement from the plasma membrane to the endoplasmic reticulum. J Biol Chem (2004) 279:45226–34.
33 . Kastaniotis AJ, Zitomer RS. Rox1 mediated repression. Oxygen dependent repression in yeast. Adv Exp Med Biol (2000) 475:185–95.[Web of Science][Medline]
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