Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA1
Seattle Biomedical Research Institute, 4 Nickerson St Suite 200, Seattle, WA 98109-1651, USA2
Department of Medicine, Santa Clara Valley Medical Center and California Institute for Medical Research, San Jose, CA 95128, USA3
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA 52242, USA4
Department of Pathology, University of Iowa, Iowa City, IA 52242, USA5
Author for correspondence: Theodore C. White. Tel: +1 206 284 8846 ext. 344. Fax: +1 206 284 0313. e-mail: tedwhite{at}u.washington.edu
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ABSTRACT |
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Keywords: pathogenic fungi, mating locus, drug resistance
Abbreviations: CHEF, contour-clamped homogeneous electric field; chr, chromosome; EtBr, ethidium bromide; SNP, single nucleotide polymorphism
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INTRODUCTION |
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A number of mechanisms have been correlated with azole resistance, including mutation and overexpression of the gene encoding the azole target enzyme, ERG11, and overexpression of the genes encoding the efflux pumps MDR1, CDR1 and CDR2 (White et al., 1998 ). However, there remains a significant portion of resistant clinical isolates in which none of the resistance mechanisms previously described can be identified (Stevens & White, 2000
).
The C. albicans mating-type-like (MTL) loci are homologous to the Saccharomyces cerevisiae mating type (MAT) loci. The MAT loci contain the transcriptional regulators MATa1, MAT1, and MAT
2 that determine the mating type (MATa or MAT
) of haploid S. cerevisiae strains (Johnson, 1995
; Herskowitz et al., 1997
). In haploid MATa strains, the MATa1 gene product has no known function. In haploid MAT
strains, Mat
1p acts as an activator of the
mating-type specific genes and Mat
2p inhibits mating-type a specific genes. In diploid S. cerevisiae, both the MATa and MAT
loci are present. In these diploid strains Mata1p and Mat
2p form a heterodimer that acts to repress haploid-specific genes.
The C. albicans MTL loci contain genes homologous to MATa1, MAT1 and MAT
2 in the same genomic arrangement as the S. cerevisiae genes (Hull & Johnson, 1999
). However, the C. albicans loci also contain genes unique to fungal mating loci, including genes encoding an oxysterol-binding protein (OBP), a poly(A) polymerase (PAP), and a phosphoinositol kinase (PIK) (Fig. 1
). A copy of each of these three genes is present in both the MTLa locus and the MTL
locus. The a and
copies of each of the three genes have only 60% identity to each other, suggesting that homologous recombination within the MTL loci is not common, unlike the rest of the genome (Hull & Johnson, 1999
). The MTL loci span approximately 9 kb and are flanked on both sides by identical sequence, suggesting that the flanking regions undergo homologous recombination like the rest of the genome (Hull & Johnson, 1999
). Most wild-type C. albicans strains are diploid and have both the MTLa and MTL
loci (Magee & Magee, 2000
).
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The current study describes a screen of clinical isolates to determine their genotype at the MTL locus. That screen demonstrated a significant correlation between homozygosity at the C. albicans MTL locus and resistance to the antifungal fluconazole. Southern analysis was used to confirm the PCR results and to demonstrate the presence of duplicate copies of the remaining MTL locus in the MTL homozygous strains.
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METHODS |
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Susceptibility testing.
Fluconazole MICs were determined by broth microdilution or macrodilution using the standardized NCCLS protocol (National Committee for Clinical Laboratory Standards, 1997 ) as described previously (Stevens & White, 2000
). Confirmation of the MICs for the MTL homozygous strains was done using E-test strips according to the manufacturers instructions (AB Biodisk, Solna, Sweden).
PCR analysis.
Oligonucleotides used for PCR in this study are listed in Table 1. PCR was performed in 50 µl reactions containing: 1xPCR buffer (50 mM KCl, 10 mM Tris/HCl, pH 8·3), 2·5 mM MgCl2, 200 µM dNTPs, 100 ng template DNA ml-1, 20 µM each primer, and 5 U Taq polymerase. PCR was performed under the following conditions in a PTC-100 programmable thermal controller (MJ Research): denaturing at 94 °C for 10 min followed by 30 cycles of 1 min denaturing at 94 °C, 2 min annealing at 55 °C, and 3 min elongation at 72 °C, followed by a final extension step at 72 °C for 10 min. PCR fragments were visualized by electrophoresis on a 1·2% agarose gel in 1xTBE (0·089 M Tris/borate, 0·089 M boric acid, 2 mM EDTA) at 100 V for 1 h and stained with ethidium bromide (EtBr).
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CHEF (contour-clamped homogeneous electric field) gels.
Gel blocks for CHEF gels were made using the following method (adapted from the method posted at http://alces.med.umn.edu/candida/methods.html). From overnight cultures, 2x108 cells were removed and washed twice in 50 mM EDTA pH 7·5. Cells were resuspended in 200 µl EDTA pH 7·5 and treated with 15 µl Zymolyase solution (50%, v/v, glycerol, 2·5 mg Zymolyase ml-1, 10 mM sodium phosphate pH 7·5). An equal volume of 1·5% low-melting-point agarose in 1xTAE (4 mM Tris/acetate, 1 mM EDTA pH 8) was added and the mixture poured into moulds. Blocks were allowed to cool at 4 °C for 30 min and were then added to a solution of 0·5 M EDTA, 10 mM Tris/HCl pH 7·5, and incubated overnight at 30 °C. Blocks were then washed three times in 0·5 M EDTA pH 9·5, treated with 2 ml of a lysis solution (2·5 mg proteinase K ml-1, 1% N-lauroylsarcosine, 0·5 M EDTA, 10 mM Tris/HCl pH 9·5) and incubated overnight at 50 °C. Blocks were washed three times with 50 mM EDTA pH 7·5 and stored for up to 3 months at 4 °C in 50 mM EDTA pH 7·5.
CHEF electrophoresis was done using a Bio-Rad CHEF Mapper with the following conditions: 0·8% agarose gel run at 14 °C at 3 V cm-1 with a 106 degree angle in 1xTAE. Running time was 50 h with a 28 min linear ramp. CHEF gels were stained with EtBr and photographed using an EagleEye II Still Video System (Stratagene). Band intensities were quantified using ImageQuant software (Molecular Dynamics).
Southern blots.
CHEF gels were treated with 0·1 M HCl for 15 min followed by three washes in water. Southern blots were then performed using standard techniques (Sambrook et al., 1989 ). Probes for labelling Southern blots were made using Prime-a-gene kits (Promega) or kinase labelling (Sambrook et al., 1989
). PCR products amplified from CAI4 DNA using the primers listed in Table 1
were used as probes for MTLa1, MTL
1, OBPa, OBP
and MTL
2. The triple probe (MTLa1-ERG11-MTL
1) was constructed by using two oligonucleotides containing 50 bp tails homologous to one of the MTL genes and 20 bp heads homologous to a region of the ERG11 gene to amplify a 150 bp double-stranded fragment that contained 50 bp of MTLa1, ERG11 and MTL
1. This fragment was radiolabelled and used to probe for those genes. Southern blots were scanned using a STORM phosphoimager (Molecular Dynamics) and quantified using ImageQuant software according to the manufacturers instructions.
Single nucleotide polymorphism (SNP) analysis.
PCR fragments from chr 5, chr 6 and chr 7 (PDE1, ARG4 and C2F7 from Table 1) were amplified from the MTL homozygous strains as previously described (Cowen et al., 1999
). These fragments were then digested with the appropriate restriction enzyme (Table 1
) in 40 µl reactions containing 1xreaction buffer, 10 µg PCR DNA, and 1 µl restriction enzyme incubated overnight at 37 °C. Digestion products were then electrophoresed in 1·5% agarose in 1xTBE at 100 V for 1 h with EtBr.
The terminator region of ERG11 on chr 5 was amplified using the primers listed in Table 1 and the conditions used to amplify the SNP fragments. This PCR fragment was sequenced (Marr et al., 1998
) using the same primers as used for PCR and screened for heterozygous polymorphisms visible as overlapping peaks on the sequencing read. Heterozygous polymorphisms were confirmed from the sequence of the complementary strand of the PCR fragment.
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RESULTS |
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Statistical analysis of the 96 strains (Table 3) indicates a strong correlation between homozygosity at the MTL locus and azole resistance (Fisher exact, P<0·0029) (Uitenbroek, 1997
). The equal distribution of MTLhom isolates, with six missing MTLa1 and six missing MTL
1, suggests that homozygosity at either locus is associated with resistance.
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However, in one of these series (FH) a switch to a homozygous MTL locus corresponded to the first significant increase in fluconazole resistance (Fig. 4
). This increase had previously been correlated with an increase in the expression of CDR1 (Marr et al., 1997
). The final isolate in this series is sensitive to fluconazole and is also heterozygous at the MTL locus (Fig. 4
) (Marr et al., 1997
). It is possible to induce azole resistance in vitro in the first sensitive isolate of this series by serially transferring the isolate in the presence of fluconazole (Marr et al., 1998
). Several of the resulting resistant isolates, induced to resistance in vitro, are also homozygous MTL
(data not shown). This correlation provides further evidence of a link between loss of MTL heterogeneity and azole resistance.
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In these analyses, a PCR fragment is amplified and digested with appropriate restriction enzymes. One of the two alternative sequences at these SNP locations contains a restriction site, the other sequence does not. This allows for rapid screening of sequence differences without direct sequencing. Short stretches of DNA from genes on chr 5 (ERG11, PDE1), chr 6 (locus C2F7), and chr 7 (ARG4) containing known SNPs (Cowen et al., 1999 ) were analysed in this way. Heterozygosity was detected by the presence of both digested and undigested bands, and would indicate the presence of at least two copies of the chromosome carrying the SNP. Homozygosity could result from either homozygous multiple copies or a single copy of the SNP. In 8 of the 12 MTLhom isolates at least one heterozygous SNP was found (Table 2
). Three of these heterozygous SNPs were on chr 5.
In addition to PCR, sequencing and Southern analysis were used to detect heterozygous SNPs. Sequencing of the noncoding region between ERG11 and THR1 revealed heterozygosity on chr 5 for two additional isolates. The heterozygosity of the ERG11 bands in Fig. 6(a) is another form of SNP analysis demonstrating that three of the 12 MTLhom strains are diploid at the ERG11 locus. The heterozygosity of the ERG11 bands in Fig. 6(a)
was only seen in strains already known to have other heterogeneous SNPs on chr 5. A total of 5 of the 12 MTLhom strains have heterogeneity of chr 5, and 10 of the 12 strains have some known heterogeneity. This suggests that most of these MTLhom strains are not haploid and provides further evidence that at least five of these strains carry two copies of chr 5.
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The MTLa1 and MTL1 bands were easily identified by their absence in the MTLhom strains. The two different higher molecular mass bands are differently sized restriction fragments of ERG11 caused by disruption of a HaeIII restriction site. All bands were of the expected size predicted by HaeIII restriction maps of the published sequences (Table 1
). The intensity of the MTL bands on three separate Northern blots was quantified and normalized to the total intensity of the ERG11 band(s). The scale was then adjusted so that the relative intensity of the CAI4 band was set to one. Although there was some variation around the mean, the relative intensities of the MTLa1 and MTL
1 bands of the MTLhom isolates were approximately twice the intensity of those bands in the control MTLhet strains (Fig. 6b
). This indicates that there are two copies of the MTLa or MTL
locus in the MTLhom strains, suggesting that the MTLhom strains are the result of gene conversion or mitotic recombination that replaced one of the MTL loci with a duplicate of the other. The presence of two ERG11 restriction fragments in some MTLhom strains, including isolates FH1 and FH5 from a serial set of isolates, suggests that the hypothetical gene conversion or mitotic recombination did not involve the ERG11 gene.
Fluconazole susceptibility is unaltered in MTL locus disruptions
It is possible that the MTL genes themselves have an effect on azole drug resistance. Gene disruptions of MTLa1, MTL1 and MTL
2 have been previously described (Hull et al., 2000
). In these gene disruptions URA3 was inserted using homologous recombination to disrupt the gene or locus of interest in the ura3 auxotrophic strain CAI4. The susceptibility of these isolates to fluconazole was measured using Etest strips (according to the manufacturers specifications) and by the standard NCCLS microbroth method (National Committee for Clinical Laboratory Standards, 1997
). The URA3-disrupted strains and ura3 auxotrophic revertants of those strains displayed no change in susceptibility compared to the parental and wild-type strains when tested on Etest strips (Table 4
). These results were verified using the NCCLS microdilution assay. However, in the NCCLS assays a subtle trend was visible (Fig. 7
). All of the strains in which MTL
1 and MTL
2 had been deleted were able to grow a small amount, even at the highest levels of fluconazole. This sort of residual growth has previously been documented as a problem in determining macrodilution MIC endpoints, although the levels seen for the MTL
1 strains would not alter the MIC of the strains.
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DISCUSSION |
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The PCR screen of 96 clinical isolates clearly showed a relationship between MTL homozygosity and azole resistance (Table 3). Although the statistical relationship between drug resistance and MTL homozygosity is significant, the presence of the sensitive MTLhom isolate P24 suggests that MTL homozygosity alone is not sufficient for resistance. This conclusion was further supported by the unchanged susceptibility of the MTL gene disruptions (Fig. 7
). These sensitive MTLhom strains may be independent of resistance, or missing functional genes necessary for MTL-related resistance, or are starting to develop resistance.
The results of the PCR screen were confirmed by additional PCR screens of other genes in the MTL loci and by the hybridization of radiolabelled MTLa1 and MTL1 genes to CHEF Southern blots (Fig. 3
). The MTLa1 and MTL
1 probes hybridized to chromosomes other than chr 5 in some of the MTLhom isolates. These extra copies were present in only a few of the MTLhom isolates and are therefore unlikely to be related to increased azole resistance in these isolates.
Further evidence of a correlation between MTL homozygosity and azole resistance comes from the corresponding increase in azole resistance seen after loss of one of the MTL loci in the FH series of isolates (Fig. 4). The final isolate was taken from post-mortem lung tissue, and thus was likely protected from fluconazole selection pressure that appears to have caused the other isolates to develop resistance, and lose the MTLa locus (Marr et al., 1997
). The gain of resistance in this series of isolates has been previously correlated with upregulation of CDR1. However in three of the five MTLhom isolates in which CDR1 levels have been analysed no upregulation of CDR1 is seen (data not shown). In fact, no known resistance mechanism is present in all of the five resistant MTLhom isolates for which we have data.
The FH series of isolates has also been shown to have a heterogeneous resistance (HetR) phenotype (Marr et al., 2001 ). A fraction of c.f.u. from HetR strains are able to grow on plates containing fluconazole, but the resulting colonies retain both azole susceptibility and the HetR phenotype. Preliminary experiments on FH1 colonies from azole-containing plates show no loss of heterozygosity at the MTL locus.
The multiple copies of chr 5 indicated by the presence of heterozygous SNPs on that chromosome (Table 2) suggest that the loss of MTL homozyosity is not due to a loss of one copy of that chromosome. Furthermore, duplicate copies of the MTL locus (Fig. 6b
), and heterozygous SNPs on chr 5 (Table 2
) suggest that the absence of one of the MTL loci is not due to deletion or chromosome loss and duplication. Given that the SNPs from chr 5 are in non-coding regions, and thus have no selection advantage, it is unlikely that these polymorphisms arose in the short time between a theoretical loss and subsequent duplication of chr 5. Homozygosity at the MTL locus could also be achieved through a gene conversion or homologous recombination that replaced the MTLa locus with a second copy of the MTL
locus or the reverse. Although this loss of heterogzygosity extends through the MTL locus, the presence of heterozygous restriction fragments in ERG11 in the FH series both before and after heterozygosity was lost at the MTL locus (Fig. 6a
) suggests that this region does not include ERG11. Mapping the size of the region of lost heterozygosity will distinguish between those two possibilities and will limit the list of genes potentially responsible for this correlation.
Changes in chromosome copy number have been linked to various morphological mutants, growth in L-sorbose and alterations in carbon and nitrogen utilization (Janbon et al., 1998 , 1999
; Rustchenko et al., 1994
). Changes in copy number of chr 4 (monosomy) and chr 3 (trisomy) have been associated with azole resistance (Perepnikhatka et al., 1999
). However, changes in copy number of chr 5 have not previously been associated with drug resistance. This study has focused on the MTL locus on chr 5, which remains disomic in these isolates. Therefore, the changes observed at the MTL locus are unlikely to be related to the changes in chromosome number observed above.
Our results suggest a number of genes that could be responsible for a correlation between MTL homozygosity and azole resistance. These possibilities can be split into two categories. The first category consists of genes that cause slight resistance when mutated in one allele and much greater resistance when the mutation is in both alleles. Selective pressure would then favour replacement of the wild-type allele with a mutant allele in a population of C. albicans grown in the presence of azole drugs. Any gene within the region of lost heterozygosity that is present in two copies could potentially fall into this category. ERG11 is the obvious example of this sort of selection, as it is the only gene related to azole resistance that lies on chr 5, and previous experiments have shown that gene conversion does occur following a point mutation in one allele of ERG11 (White, 1997b ; Franz et al., 1998
). However, the presence of heterozygous SNPs in the ERG11 coding region and terminator in 5 of the 12 MTLhom suggests that there has been no recent loss of heterozygosity in ERG11 in these isolates and therefore that ERG11 is not the gene responsible for the correlation between MTL homozygosity and resistance. Other genes that could potentially fall into this category include the OBP and PIK genes in the MTL loci. Homologues of the OBP genes in particular have been shown to play a role in ergosterol homeostasis in S. cerevisiae (Beh et al., 2001
).
The other category of possibilities is a general resistance effect that results from the loss of one of the MTL specific genes. Of particular interest are the MTLa1 and MTL2 genes. The homologues of the products of these genes in S. cerevisiae form a heterodimeric transcriptional regulator (Herskowitz et al., 1997
). It therefore follows that if a functionally similar heterodimer forms in C. albicans, loss of one of those genes would lead to altered levels of a number of genes, possibly including those involved in azole resistance. Although the gene disruptions of the MTL loci caused little change in azole susceptibility (Table 4
), the laboratory strain in which the gene disruptions were performed may have a nonfunctional copy of a gene that contributes to resistance.
Future experiments will determine which of these models is correct. These experiments include characterizing the MTLhom strains further and determining the mechanism by which they develop resistance decreased import, increased export, increased resistance of the Erg11p in these isolates, or some other undescribed mechanism.
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ACKNOWLEDGEMENTS |
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Received 8 October 2001;
revised 7 December 2001;
accepted 14 December 2001.