1 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; 2 Institut für Mikrobiologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
Received 10 January 2005; returned 11 February 2005; revised 28 February 2005; accepted 1 March 2005
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Abstract |
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Methods: We disrupted the ERG1 gene of C. albicans, which encodes squalene epoxidase (EC 1.14.99.7). Since the disruption of both alleles of ERG1 was lethal, the second allele of a heterozygous disruptant was placed under the control of a regulable promoter, MET3p, which is repressed by methionine and cysteine.
Results: The reverse-phase HPLC analysis of sterol, extracted from the conditional mutant strain, showed a total lack of ergosterol and instead accumulation of squalene. This imbalance in sterol composition led to defects in growth and increased susceptibilities to drugs including fluconazole, ketoconazole, cycloheximide, nystatin, amphotericin B and terbinafine. Reduced drug efflux activity of the erg1 mutant was associated with poor surface localization of Cdr1p, suggesting that enhanced passive diffusion and reduced efflux mediated by the ABC (ATP binding cassette) transporter Cdr1p increases drug susceptibility. Additionally, conditional erg1 mutant strains were unable to form hyphae in various media.
Conclusions: Taken together, our results demonstrate that the absence of ergosterol, which is one of the constituents of membrane microdomains (rafts), has a direct effect on drug susceptibility and morphogenesis of C. albicans.
Keywords: regulable promoter , hyphae , drug resistance , Cdr1p , ergosterol , dimorphism
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Introduction |
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The important role of lipids in drug susceptibilities of Candida cells has become apparent from recent studies.57 Azole-resistant C. albicans isolates exhibit altered membrane phospholipid and sterol composition.6,810 We and others have observed that ABC transporters Cdr1p of C. albicans and Pdr5p of Saccharomyces cerevisiae are particularly sensitive to changes in lipid composition where functions mediated by these drug extrusion pump proteins are affected.5,11,12 Taken together, it appears that the associated changes in membrane lipid composition (phospholipid/ergosterol), its order (fluidity), and asymmetry could be important determinants of drug susceptibilities of yeast cells.6,7
Among various classes of lipids in C. albicans, membrane ergosterol is an important constituent, which is also the target of common antifungals like polyenes and azoles.1315 Ergosterol is also responsible for membrane rigidity, stability and resistance to physical stresses. In addition, ergosterol modulates membrane fluidity, permeability and the activities of membrane-bound enzymes.16 Interestingly, the action of antifungals is affected by the changes in membrane lipid composition in general and by ergosterol in particular.12,16 Because of the relationship between drug resistance and membrane ergosterol composition, many genes of the ergosterol biosynthetic pathway of C. albicans such as ERG3, ERG6, ERG11, ERG24 and ERG26 etc. have been analysed.14,1719
ERG1 encodes squalene epoxidase (EC 1.14.99.7), which is a FAD-containing monooxygenase that converts squalene into 2,3-oxidosqualene.13,20,21 Squalene epoxidase plays a key role in the synthesis of essential sterol compounds, hence homozygous disruption of ERG1 was found to have deleterious effects in yeast cells.22,23 In order to explore the direct involvement of ergosterol in morphogenesis and drug susceptibility of C. albicans, in this study, we disrupted one allele of ERG1 and expressed the second allele under the control of the regulable MET3 promoter.24,25 We show that conditional suppression of ERG1 of C. albicans increases its susceptibility to drugs and leads to defects in hyphae formation. The enhanced susceptibility of the conditional erg1 mutant to various drugs is linked to increased passive diffusion and reduced efflux of drugs mediated by ABC transporter Cdr1p, which as a result of imbalance in sterol composition was poorly localized to the plasma membrane.
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Materials and methods |
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Media chemicals were obtained from Difco (Detroit, MI, USA) and HiMedia (Mumbai, India). The drugs cycloheximide, terbinafine, nystatin, amphotericin B, rhodamine-6G and protease inhibitors (PMSF, leupeptin, pepstatin A, aprotinin) were obtained from Sigma Chemical Co. (St Louis, MO, USA). Nourseothricin was obtained from Werner Bioagents (Jena, Germany). Ranbaxy Laboratories (New Delhi, India) kindly provided fluconazole, while ketoconazole was a gift from Dupont (Wilmington, DE, USA).
Bacterial and yeast strains and growth media
Plasmids were maintained in Escherichia coli DH 5. E. coli was cultured in LuriaBertani medium (Difco, BD Biosciences, NJ, USA), to which ampicillin was added (100 mg/L). C. albicans CAI4, CAF2-1 and the heterozygous disruptant strains were grown in Yeast Extract Peptone Dextrose (YEPD) broth or synthetic defined (SD) medium (Bio 101, Vista, CA, USA).25
To repress the MET3 promoter, the conditional ERG1 mutants were grown in SD medium supplemented with 0.5 mM each of methionine and cysteine (M/C).
Deletion of ERG1 and chromosomal integration of MET3p-ERG1
ERG1 of C. albicans CAI4 was disrupted by employing the URA-Blaster method.26,27
A cassette for the disruption of ERG1 was constructed in several steps. Firstly, 690 bp of the 5' sequences flanking the start of the CaERG1 ORF were amplified using primers ERG1disA and ERG1disB (5'-CTAGAGCTCGGATCCGTGTTTACTCATATG-3' and 5'-CTAGAGCTCGGATCCGGACTGGCACTATTCTC-3': bold, regions of homology; italics, BamHI). Similarly, 761 bp of the 3' untranslated sequences were amplified by primers ERG1disC and ERG1disD (5'-CTAGAGCTCTGCAGATTGTAAGATAGAGAG-3' and 5'-CTAGAGCTCTGCAGGTTCTTTCGTCCC-3': bold, regions of homology; italics, PstI). PCR fragments were sub-cloned into pUC18, which resulted in plasmids pSKM3 and pSKM6, respectively. pSKM3 was digested with BamHI and cloned into the BglII site of pUC18. The PstI fragment of pSKM6 was inserted into the PstI site of the resulting plasmid pSKM53. A plasmid with ERG1 flanking sequences, in the correct orientation, was obtained (pSKM54). Its SacISphI fragment containing the ERG1 disruption cassette was used to obtain heterozygous strain E4 (ERG1/erg1
::hisG-URA3-hisG) by transformation. URA3 was looped out from
E4 using 5-fluoroorotic acid (1 mg/mL) and 25 mg/L uridine to obtain
E4.2 (ERG1/erg1
::hisG).
To place ERG1 under the control of the MET3 promoter, we followed a previously described strategy.25
First, a fragment of 543 bp corresponding to the 5'-end of the ERG1 ORF was amplified by PCR using the primers ErgN-term (5'-GCACACCAATAACTGTG-3') and ErgStpBglIIHindIII (5'-CTTAAGCTTAGATCTAGTCTCTCTATCTTAC-3') and cloned into pUC18, resulting in plasmid pSKM12. The BamHI fragment of pSKM12 was inserted into the single BamHI site of pCaDis, downstream of the MET3 promoter.24
The resulting plasmid pSKM14 was linearized by NcoI (which cuts within the ERG1 fragment) and was used to transform the heterozygous strain
E4.2 (ERG1/erg1
::hisG) to obtain the conditional mutant
E4.2.7 (MET3p-ERG1/erg1
::hisG). Integration of heterozygous and conditional strains was checked by Southern blots.
Tagging of Cdr1p with GFP
To tag CDR1-ORF with GFP, its stop codon was mutated into a BamHI site. For this, 250 bp of the C-terminal CDR1 ORF was PCR-amplified using primers CDR29F and CDR30R (5'-ATTTGGTACCATACATTAAATTTGCTGGTGGG-3' and 5'-GTTTGGATCCTTTCTTATTTTTTTTCTCTCTGTTACCC-3': bold, regions of homology; italics, KpnI and BamHI). The PCR amplicon was digested with KpnIBamHI (sites introduced into forward and reverse primers during synthesis). GFP along with the ACT1 termination sequence and the dominant selection marker gene SAT1 were taken out by digesting vector pADH1G3 with BamHIPstI.28
The KpnIPstI-digested vector backbone of pCPL51 (2.9 kb) was ligated with KpnIBamHI-digested C-terminal CDR1 along with BamHIPstI-digested GFP-SAT1 fragment to generate the final plasmid pCPG2. Its 3.5 kb KpnISacII fragment having CT-CDR1-GFP-SAT1-3'UTR was used to integrate CAF2-1 and E4.2.7 at the CDR1 locus. The transformants were selected on YEPD plates containing 200 mg/L of nourseothricin.28
Transformants were designated as CAF2-1CDR1GFP and
E4.2.7-CDR1GFP for further study.
Growth of the mutant strains
C. albicans strains were grown for 20 h in SD medium with 0.5 mM methionine and 0.5 mM cysteine and their growth was monitored at 600 nm after regular time intervals. The growth of the conditional strains was found to be maximally suppressed at 14 h after the addition of M/C, and cells at this time point were collected and used for further analysis.
Identification of sterols by HPLC
Ergosterol formation and squalene accumulation were determined by reverse-phase HPLC using an HPLC system (Biologic Duo-Flow, Bio-Rad, UK). Sterols were extracted by the alcoholic KOH method7 and reverse-phase chromatography was carried out using a C-18 column fitted with a C-18 guard column. Elution was carried out using the previously described gradient program.7,29 Samples were injected as solutions in 95% ethanol. Elution of compounds was automatically monitored by absorption at 214, 230, 260 and 282 nm. Ergosterol and squalene were used as standards (from Sigma) for comparison of their respective retention times.
Drug susceptibility testing in C. albicans strains
As a result of the exhaustion of methionine and cysteine after 18 h in the growth medium, it was not possible to determine the MICs of drugs and therefore only spot assays were performed for testing drug susceptibilities of the conditional erg1 mutant strain. In spot assays, 5 µL of 5-fold serial dilutions of each yeast culture (A600=0.1) was spotted on to SD plates supplemented with and without 0.5 mM methionine and 0.5 mM cysteine, in the absence (control) and in the presence of the drugs. Growth differences were recorded following incubation of the plates for 2 days at 30°C.
Passive diffusion and rhodamine 6G (R6G) efflux
Passive diffusion and efflux of fluorescent R6G was determined using a protocol described previously,6,7 except that SD medium supplemented with M/C was used for culturing cells. Cells were pelleted down and a 2% cell suspension was made in de-energization phosphate-buffered saline (PBS) containing 5 mM 2,4-dinitrophenol and 5 mM deoxy-D-glucose and incubated for 2 h at 30°C.6,7,12 Cells were then washed and incubated in PBS containing 10 µM R6G. For passive diffusion, a 1 mL aliquot was taken out at different time intervals, centrifuged at 9000 g for 2 min and the optical density of the supernatant was determined at 527 nm.6,7 Efflux was initiated by the addition of 1 mole of glucose and an aliquot of 1 mL was taken out at regular intervals for measuring optical density at 527 nm.
Plasma membrane preparation and western blotting
Plasma membranes (PM) were prepared as described previously,30 except that SD medium without (control) and with M/C was used for cell culture. Western blotting was done with anti-Cdr1p and anti-Pma1p antibodies.30 PM proteins were separated on an 8% SDS/polyacrylamide gel and electroblotted onto a nitrocellulose membrane and incubated with rabbit anti-Cdr1p antibody (diluted 1:500) and rabbit anti-Pma1p antibody (diluted 1:10 000). Proteins on immunoblots were detected using the chemiluminescence assay system (ECL kit; Amersham Biosciences).30
GFP fluorescence microscopy
The cells were grown in SD medium with and without M/C, placed on glass slides and directly viewed under a x100 oil immersion objective of a confocal microscope (Radiance 2100, AGR, 3Q/BLD, Bio-Rad, UK).
Hyphal morphogenesis in mutant strains
Strains were grown for 5 days at 37°C on solid Lee's and Spider media.25 Additionally, hyphal-inducing liquid media supplemented with 15% serum and N-acetyl glucosamine supplemented with 0.50 mM of M/C were also used.31 Following incubation, the colonies and cells were checked for hyphae formation under a Zeiss Axioscope microscope.
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Results and discussion |
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For the disruption of ERG1, which encodes squalene epoxidase, a key enzyme of ergosterol biosynthesis, C. albicans sequence data were obtained from the Stanford Genome Technology Centre (http://www.sequence.stanford.edu/group/candida). The deduced C. albicans Erg1 protein contains 496 residues, with a predicted molecular mass of 55.3 kDa.20
The genomic region of ERG1 was isolated by PCR using genomic DNA of the CAI4 strain. A SacISphI fragment from plasmid pSKM54 containing an ERG1 disruption cassette was used to transform the CAI4 strain to obtain heterozygous strain E4 (ERG1/erg1
::hisG-URA3-hisG). Heterozygous ERG1/erg1 strains were generated without any difficulty but construction of the homozygous erg1/erg1 strain using the same hisG-URA3-hisG blaster cassette repeatedly failed. This suggested that probably similar to the ScERG1 gene of S. cerevisiae, CaERG1 of C. albicans is an essential gene.23
To reconfirm this, we put the second allele under the control of the MET3 regulable promoter, which is repressed by M/C.24,25
The cells with one ERG1 allele under the control of the regulable promoter were designated as E4.2.7 (MET3p-ERG1/erg1
::hisG) (Figure 1a). Correct integrations in heterozygous and conditional strains were checked by Southern blot, as shown in Figure 1(b). Two independent clones were picked and analysed simultaneously for the phenotypes described below. Pairs of isogenic mutant strains were identical in all phenotypes tested.
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The conditional mutant strain E4.2.7 (MET3p-ERG1/erg1
:: hisG) was grown in liquid SD medium with or without M/C. We observed that 0.5 mM of M/C was enough to considerably suppress the growth of the conditional mutant
E4.2.7, while growth of the wild-type CAF2-1 and heterozygous strain
E4 remained almost unaffected under similar repressing conditions (Figure 1c). However, growth suppression ceased after 24 h and conditional mutants with suppressor were found to regain their growth. Of note, strain
E4.2.7, when streaked on SD plates containing 0.5 mM M/C, continued to show suppression of growth and no regrowth was observed. The regrowth of conditional strain at 24 h could be because of the leakiness of the MET3 promoter as was reported previously,32
or partly could be due to exhaustion of M/C.
ERG1 conditional mutant strain lacks ergosterol
Mutants of the ergosterol biosynthetic pathway do not produce ergosterol and utilize sterol intermediates to compensate for the loss of ergosterol.5,8
To confirm the status of ergosterol levels in the erg1 mutant strain, reverse-phase HPLC was employed.7,29
The HPLC analyses of extracted sterols revealed that ergosterol levels in the heterozygous strain E4 were lower compared with the wild-type CAF2-1. Interestingly, the conditional disruptant strain (
E4.2.7), after growth in the presence of M/C, showed no detectable characteristic ergosterol peak with a retention time of 40 min, but instead showed a high peak of squalene at a retention time of 50 min (Figure 2). Thus, HPLC analysis revealed that
E4.2.7 strain, which conditionally lacks functional squalene epoxidase (Erg1p), is unable to synthesize ergosterol and instead accumulates squalene (Figure 2). The lack of ergosterol in the conditional strain was also confirmed by spectrophotometric analysis of extracted sterols (data not shown).7
Both the quantitative methods thus confirmed that the conditional strain in the presence of M/C does not synthesize ergosterol and instead accumulates squalene.
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In view of the fact that alterations in lipid composition affect drug susceptibilities,6,7,11,33
we examined whether the depletion of ergosterol and concomitant accumulation of squalene affected drug susceptibilities of conditional disruptant cells. Interestingly, under repressing conditions in serial dilution spot assays, the E4.2.7 cells were hypersusceptible to all tested drugs (fluconazole, cycloheximide, ketoconazole, amphotericin B, nystatin and terbinafine) compared with CAF2-1 and heterozygous
E4.2 (Figure 3). The increased susceptibility of
E4.2.7 conditional mutant cells to terbinafine needs special mention. Terbinafine is known to target ERG1, therefore, its conditional disruption, as in
E4.2.7 cells, should result in resistance rather that hypersusceptibility. However, our observations support previous reports in Candida glabrata,22
where an erg1 defect led to increased susceptibility to terbinafine. These results imply that various unidentified factors could also modulate the response of fungal cells to this allylamine.13,23,34
Interestingly, the erg1 conditional strain
E4.2.7 became susceptible to polyenes, nystatin and amphotericin B, even in the complete absence of detectable ergosterol (Figure 3). This finding was unexpected, because polyenes specifically interact with ergosterol and hence erg1 mutants lacking ergosterol are expected to be resistant to these drugs. Our results support increasing evidence suggesting that membrane lipids other than ergosterol could also influence polyene susceptibility.13,23,34
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Both the entry (passive diffusion) and exit (efflux) of drugs affect the drug susceptibilities of Candida cells.6,7
We therefore examined this aspect by measuring passive diffusion and energy-dependent efflux of R6G, which is a substrate of Cdr1p of C. albicans.7
The conditional erg1 knockout strain E4.2.7, in the presence of M/C, displayed decreased amounts of extracellular R6G in de-energized cells, suggesting increased passive diffusion of the substrate (Figure 4). To determine active drug efflux, the fluorescent compound R6G was allowed to equilibrate in de-energized Candida mutant cells by passive diffusion and the energy-dependent extrusion of R6G was then initiated by the addition of glucose. As depicted in Figure 4, reduction in glucose-induced efflux, indicated by a decrease in extracellular concentration of R6G, was seen in conditional mutant strain
E4.2.7 (70% reduction in efflux of R6G at 25 min after the addition of glucose, compared with the CAF2-1 cells; see inset of Figure 4). Thus, the increase in passive diffusion and reduced ability to efflux, both probably contribute to hypersusceptibility of the conditional erg1 mutant. Here we would like to mention that the conditional mutant without the suppressors, which is similar to the heterozygous strain, showed similar growth to the wild-type cells. This erg1/ERG1 conditional strain without M/C could already show the difference in its ability to efflux R6G. This gene dosage-dependent reduction in efflux in ERG1/erg1 implies that efflux changes are not growth-dependent. Thus addition of M/C which further reduces the efflux of the erg1/erg1 strain is due to suppression of the second allele and not due to the slow growth.
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We have previously observed that the ABC transporter Cdr1p is selectively sensitive to changes in lipid composition.6,7
It is thus possible that the observed impaired efflux in the conditional mutant strain could be due to poor functionality of Cdr1p. Our immunoblot results revealed that in E4.2.7 cells, which were collected after 14 h of M/C addition in SD medium, the expression of Cdr1p in PM was decreased compared with PM isolated from the wild-type strain (Figure 5a).
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Hyphal morphogenesis requires wild-type ERG1 expression levels
The availability of a conditional strain, in which ERG1 expression could be suppressed, provided an opportunity to examine if it affects morphogenesis of C. albicans. The ability of erg1 disruptants to form hyphae was checked on solid Lee's and Spider media. Additionally, hyphal-inducing liquid media supplemented with 15% serum and N-acetyl glucosamine supplemented with 0.50 mM of M/C were also used.31
As compared with CAF2-1, the heterozygous strain E4 was found to have reduced formation of hyphae, while the conditional disruptant strain
E4.2.7 (erg1/erg1) appeared as colonies lacking filaments and thus had completely lost its ability to form hyphae under the conditions tested (Figure 6a and b). The inability of the conditional disruptant strain to form hyphae in all the tested media suggests that wild-type Erg1p-levels are critical for morphogenesis of C. albicans (Figure 6). Of note, both media used for hyphal induction already contained M/C and complex sources of nitrogen, therefore, no additional repressor to the hyphal media was necessary to partially suppress ERG1 expression, while still permitting growth.25
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In conclusion, we demonstrate that any imbalance in sterol composition severely increases drug susceptibility of C. albicans cells. The complete lack of ergosterol in the conditional erg1 mutant and/or accumulation of squalene therein affect functioning of the ABC drug extrusion pump protein Cdr1p mainly because of its poor surface localization. There have been previous reports describing the role of ergosterol in the morphogenesis of Candida,35,36 but in this study, we show a direct link between sterol composition and the ability of Candida cells to form hyphae. In this context, it is important to refer to a recent report, where the presence of polarized membrane domains, rich in ergosterol and sphingolipid in C. albicans, was demonstrated.37 Both ergosterol and sphingolipids are two major constituents of membrane microdomains (membrane rafts) and blocking synthesis of either of the two results in defects in hyphae formation and drug susceptibilities.7 Considering recent reports, it appears that Cdr1p of C. albicans may preferentially be associated with raft microdomains6,7 that remain to be demonstrated experimentally.
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Footnotes |
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Present address. Department of Biochemistry, RWJMS Research Building, UMDNJ, 683 Hoes Lane West, Piscataway, NJ 08854, USA.
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Acknowledgements |
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