. 2003 Apr;47(4):1207–1212. doi:10.1128/AAC.47.4.1207-1212.2003

Drug Resistance Is Not Directly Affected by Mating Type Locus Zygosity inCandida albicans

Claude Pujol¹,Shawn A Messer²,Michael Pfaller²,David R Soll^1,^*

Departments of Biological Sciences,¹ Pathology, The University of Iowa, Iowa City, Iowa 52242²

Corresponding author. Mailing address: 302 BBE, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-1117. Fax: (319) 335-2772. E-mail:david-soll@uiowa.edu.

Received 2002 Nov 7; Revised 2002 Dec 13; Accepted 2003 Jan 24.

PMC Copyright notice

PMCID: PMC152535 PMID:12654648

Abstract

Recently, evidence was presented that in a collection of fluconazole-resistant strains ofCandida albicans there was a much higher proportion of homozygotes for the mating type locus (MTL) than in a collection of fluconazole-sensitive isolates, suggesting the possibility that when cells becomeMTL homozygous they acquire intrinsic drug resistance. To investigate this possibility, an opposite strategy was employed. First, drug susceptibility was measured in a collection of isolates selected forMTL homozygosity. The majority of these isolates had not been exposed to antifungal drugs. Second, the level of drug susceptibility was compared between spontaneously generatedMTL-homozygous progeny and theirMTL-heterozygous parent strains which had not been exposed to antifungal drugs. The results demonstrate that naturally occurringMTL-homozygous strains are not intrinsically more drug resistant, supporting the hypotheses that either the higher incidence ofMTL homozygosity previously demonstrated among fluconazole-resistant isolates involved associated homozygosity of a drug resistance gene linked to theMTL locus, or thatMTL-homozygous strains may be better at developing drug resistance upon exposure to the drug thanMTL-heterozygous strains. Furthermore, the results demonstrate that a switch by anMTL-homozygous strain from the white to opaque phenotype, the latter functioning as the facilitator of mating, does not notably alter drug susceptibility.

The great majority ofCandida albicans strains are heterozygous at the mating type-like locusMTL, carryingMTLa1 on one chromosome andMTLα1 andMTLα2 on the homologous chromosome (7,14). Fusion has been demonstrated to occur both in vivo (8) and in vitro (16,17) between engineered homozygousMTLa (a/−) andMTLα (α/−) cells, but not between parentalMTL-heterozygous (a/α) cells. Recently, Miller and Johnson (17) discovered an extraordinary and unexpected relationship between mating and white-opaque switching. They demonstrated that while cells of theMTL-heterozygous parent strain CAI4 (a/α) did not undergo white-opaque switching, derivative homozygousMTLa and homozygousMTLα cells underwent white-opaque switching at high frequency, and that when homozygous cells were in the opaque phase, the efficiency of mating increased 10⁶-fold. Lockhart et al. (14) subsequently demonstrated that all white-opaque switchers obtained from a collection of clinical isolates wereMTL homozygous and, conversely, that the majority ofMTL homozygotes obtained from a collection of clinical isolates underwent white-opaque switching. Lockhart et al. (15) subsequently demonstrated that fusion occurred only between naturally occurring homozygousMTLa and homozygousMTLα strains in the opaque phase. Finally, Lockhart et al. (15) described for the first time the cell biology ofC. albicans mating, including shmooing, chemotropism of conjugation tubes, fusion of tubes, nuclear association, vacuole expansion and nuclear separation in the conjugation bridge, asynchronous nuclear division in the zygote, daughter cell growth, nuclear migration in the daughter cell, septation, and daughter cell budding.

Recently, Rustad et al. (27) presented evidence suggesting a link between homozygosity at theMTL locus and fluconazole resistance inC. albicans. An analysis of 46 fluconazole-sensitive and 50 fluconazole-resistant strains revealed that while only 2% of the former were homozygous for theMTL locus (i.e., eithera/a or α/α), 22% of the latter were homozygous. Rustad et al. (27) hypothesized that the association of fluconazole resistance andMTL homozygosity could be due to two possible mechanisms. First, drug resistance could be the direct result of the loss of eitherMTLa1 orMTLα1/MTLα2 (i.e., the direct result ofMTL homozygosity), which in turn suggests thatMTL-homozygous strains may be intrinsically resistant. Alternatively, they hypothesized that drug resistance could be the result of homozygosity of an allele linked toMTL on chromosome 5, through mitotic crossing over. Rustad et al. (27) demonstrated that homozygousMTLa and homozygousMTLα cells, engineered from the heterozygousMTLa/α strain CAI4 by Hull et al. (8), were as susceptible to fluconazole as the parentMTL-heterozygous strain, which supported the hypothesis that the high level ofMTL homozygosity among fluconazole-resistant strains was not due to the immediate acquisition of drug resistance throughMTL alleles becoming homozygous without drug exposure.

To investigate further the relationship between fluconazole resistance andMTL homozygosity, we have used a strategy opposite that of Rustad et al. (27). Rather than measure the proportion ofMTL homozygotes among identified drug-resistant strains, we measured drug susceptibility among identifiedMTL-homozygous strains, the majority of which were collected from patients prior to drug therapy. In addition, we tested the susceptibility of theMTL-homozygous progeny of threeMTL-heterozygous strains that spontaneously formedMTL-homozygous progeny at high frequency. If resistance is directly associated with the loss of eitherMTLa1 orMTLα1/MTLα2, thenMTL homozygotes randomly selected from a large collection of clinical isolates should be intrinsically drug resistant without exposure to drugs, andMTL homozygotes spontaneously generated by naturally occurring, unstableMTL heterozygotes should be more drug resistant than their heterozygous parent strains without exposure to drugs. The results presented here demonstrate that in the absence of previous antifungal drug exposure, naturally occurringMTL-homozygous strains ofC. albicans are not intrinsically more drug resistant.

MATERIALS AND METHODS

C. albicans isolates.

A general collection of over 300 clinical isolates representing the five major clades distinguished by the complex DNA fingerprinting probe Ca3 (5,14,23,25) were tested forMTL zygosity by PCR methods (14,15). A total of 16MTL-homozygous strains were identified, including 7MTLa-homozygous and 9MTLα-homozygous strains. The majority of isolates were previously described (14). Two additional isolates,P37037 andP37039, were both collected from healthy individuals in Wisconsin and New Jersey, respectively. To this collection was added strain WO-1, which isMTLα homozygous. This population represents a mixed collection of isolates from the United States (nine isolates), Canada (one isolate), and South Africa (five isolates) from diverse clinical origins (14). With the exception of 19F and WO-1, these isolates were not exposed to any antifungal agent prior to collection. 19F was exposed to terconazole, and WO-1 was exposed to 5-fluorcytosine and amphotericin B prior to collection. Isolates were stored in 20% glycerol at 80°C prior to experimental use.

MIC testing.

Antifungal susceptibility testing was performed by the reference broth microdilution method as described in the National Committee for Clinical Laboratory Standards document M27-A (18). Caspofungin (Merck Research Laboratories, Rahway, N.J.), amphotericin B (Sigma, St. Louis, Mo.), flucytosine (5-FC; Sigma), fluconazole (Pfizer Inc., New York, N.Y.), voriconazole (Pfizer), itraconazole (Janssen, Beerse, Belgium), posaconazole (Schering-Plough, Kenilworth, N.J.), and ravuconazole (Bristol-Myers Squibb, Wallingford, Conn.) were obtained from their respective manufacturers. Serial dilutions were made in RPMI 1640 broth medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid. Microdilution trays were prepared in a single lot as previously described (21), with the addition of an array of caspofungin at final dilutions of 0.007 to 8 μg/ml. The trays were stored at −70°C prior to use.

Prior to testing, each isolate was passaged on supplemented Lee's agar medium (4,11) to assess colony phenotypes and once on potato dextrose agar (Remel, Lenexa, Kans.) to ensure optimal growth characteristics. One hundred microliters of a suspension of cells varying between 0.5 × 10³ and 2.5 × 10³ cells per ml was added to each well of a microdilution tray. The trays were incubated in air at 35°C, and MIC endpoints were read after 48 h. Drug- and yeast-free controls were included in each tray. Following incubation, the growth in each well was compared with that in the control wells. The MICs of each of the triazoles, caspofungin, and 5-FC were defined as the lowest concentration resulting in approximately 50% growth inhibition. The MIC of amphotericin B was defined as the lowest concentration resulting in 100% growth inhibition. Quality control of the susceptibility assays was performed on the reference isolatesCandida parasilosis ATCC 22091 andCandida krusei ATCC 6258 (3,18).

DNA fingerprinting.

The complex DNA fingerprinting probe Ca3 (1,13,24,28) was used to confirm the identity of the progeny isolates from the parental strains P37037par and P37039par and to exclude the possibility of contaminations. DNA was extracted from cells (31), digested withEcoRI, and electrophoresed in a 0.8% agarose gel at 50 V according to methods previously described (12,25,32,34). The DNA was then transferred to a Hybond N⁺ membrane (Amersham, Piscataway, N.J.) by capillary blotting. Prehybridization and hybridization with³²P-labeled Ca3 probe were performed as previously described (32). Southern blots were then autoradiographed.

PCR analysis of theMTL locus.

The PCR protocol used to analyze theMTL locus was previously described (14). The presence ofMTLa was ascertained by amplifyingMTLa1 and the associated geneOBPa (7). The presence ofMTLα was ascertained by amplifyingMTLα1 andMTLα2 and the associated geneOBPα.MTLα1,MTLα2, andMTLa1 are theC. albicans homologues of theSaccharomyces cerevisiae mating type genesMATα1,MATα2, andMATa1, respectively (7).

RESULTS

Drug susceptibility ofa and α cells.

To test whether naturally occurringMTL-homozygous strains are more resistant to antifungal drugs than naturally occurringMTL-heterozygous strains, we first compared the susceptibility of 7a strains and 10 α strains identified in a large collection of clinical isolates with the susceptibility of an independent set of unselected isolates from two large collections of naturally occurring isolates previously analyzed (6,22). Since Lockhart et al. recently demonstrated that approximately 97% of naturally occurringC. albicans isolates are heterozygous at theMTL locus (14), we assumed here that the large collections previously analyzed for drug susceptibility were predominantlyMTL heterozygous. In Table1, the MICs of fluconazole, ravuconazole, posaconazole, voriconazole, itraconazole, caspofungin, amphotericin B, and 5-FC are presented for each of the 17 homozygous strains. In addition, the MIC₅₀ (the concentration that inhibited 50% of isolates) and MIC₉₀ (the concentration that inhibited 90% of isolates) of the entire collection of 17MTL-homozygous strains and of the previously studied collections of predominantlyMTL-heterozygous strains are provided in Table1.

TABLE 1.

Antifungal susceptibility of 17C. albicans strains homozygous at the mating type-like locus (MTL)

MTL genotype	Strain	MIC (μg/ml)^a
MTL genotype	Strain	Flu.	Ravu.	Posa.	Vori.	Itra.	Caspo.	Ampho. B	5-FC
a	L26	2.00	0.060	0.120	0.030	0.500	0.12	1.0	2.00
	12C	0.25	≤0.007	0.015	≤0.007	0.030	0.06	0.5	0.25
	G106	0.50	≤0.007	0.030	≤0.007	0.120	0.06	1.0	0.25
	OKP90	≤0.12	≤0.007	0.015	≤0.007	0.030	0.12	1.0	16.00
	P60	0.25	≤0.007	0.015	≤0.007	0.060	0.06	1.0	0.25
	P37005	0.25	≤0.007	0.030	≤0.007	0.060	0.06	1.0	1.00
	P75063	0.25	≤0.007	0.015	≤0.007	0.030	0.12	1.0	0.12
α	WO-1	≤0.12	≤0.007	0.030	0.015	0.060	0.12	1.0	0.12
	19F	0.25	≤0.007	0.015	≤0.007	0.030	0.12	1.0	≥64.00
	GC75	≤0.12	≤0.007	≤0.007	≤0.007	0.030	0.25	1.0	0.25
	P87	0.25	≤0.007	0.030	≤0.007	0.060	0.12	1.0	0.12
	P37035	0.50	0.015	0.015	≤0.007	0.060	0.06	1.0	0.25
	P37037	0.25	≤0.007	0.015	≤0.007	0.030	0.25	1.0	0.25
	P37039	≤0.12	≤0.007	0.015	≤0.007	0.030	0.06	1.0	0.25
	P57072	0.25	≤0.007	≤0.007	≤0.007	0.015	0.06	1.0	0.12
	P78048	0.25	≤0.007	0.015	≤0.007	0.030	0.06	1.0	0.25
	P80001	0.50	≤0.007	0.030	≤0.007	0.120	0.06	1.0	0.12
All isolates
MIC₅₀^b		0.25	≤0.007	0.015	≤0.007	0.030	0.06	1.0	0.25
MIC₉₀^b		0.50	≤0.007	0.030	≤0.007	0.120	0.12	1.0	2.00
a/α (97%)^c
MIC₅₀^b		0.25	≤0.007	0.015	≤0.007	0.030	0.12	1.0	0.25
MIC₉₀^b		0.50	0.015	0.060	0.015	0.060	0.25	1.0	1.00

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Flu., fluconazole; Ravu., ravuconazole; Posa., posaconazole; Vori., voriconazole; Itra., itraconazole; Caspo., caspofungin; Ampho.B, amphotericin B.

The MIC₅₀ and MIC₉₀ values represent the concentration of each antifungal agent that inhibited 50% and 90%, respectively, of the tested isolates.

Data were obtained from Pfaller et al. (22) for a collection of 486 isolates ofC. albicans tested for caspofungin and from Diekema et al. (6) for a collection of 148 isolates ofC. albicans tested for the remaining antifungals. The collections used as controls were assumed to be 97%a/α based on the results of Lockhart et al. (14).

Of the 17MTL-homozygous strains tested, only one strain, L26, exhibited increased resistance to fluconazole (Table1). This strain also exhibited increased resistance to the other tested azoles and a slight increase in 5-FC resistance, but no resistance to caspofungin or amphotericin B. Two strains, OKP90 and 19F, exhibited increased resistance to 5-FC but normal susceptibility to all other drugs. With the exception of resistance to 5-FC observed for 19F, the twoMTL-homozygous strains that had been exposed to drug therapy prior to collection, strains WO-1 and 19F, exhibited no increases in resistance to the tested drugs. These results suggest that becoming homozygous in and of itself is not sufficient to confer drug resistance inC. albicans.

Drug susceptibility of spontaneousMTL-homozygous progeny ofMTL-heterozygous strains.

We next tested whetherMTL heterozygotes gave rise to drug-resistantMTL-homozygous progeny. In an analysis of 30MTL-heterozygous isolates, 3 were identified that formed opaque-phase sectors at high frequency. Since white-opaque switching is suppressed inMTL-heterozygous strains (14,17), it appeared likely that these particular strains gave rise spontaneously toMTL-homozygous progeny at high frequency. To test this possibility, randomly selected progeny of the three such strains were tested forMTL zygosity by PCR analysis of mating type genes. For strainsP37037 andP37039,MTLa/α andMTLα progeny were identified and, for strainP75063,MTLa/α andMTLa progeny were identified (Fig.1). In Fig.2A, PCR amplification patterns ofMTLa1 andMTLα2 are presented for theMTL-heterozygous parental strains P37037par and P37039par and bothMTL-heterozygous andMTL-homozygous progeny of each. A similar analysis of strainP75063 was previously reported (14). To demonstrate thatMTL-heterozygous andMTL-homozygous isolates were progeny of the respective parent strains and not contaminants, isolates were DNA fingerprinted by Southern blot hybridization with the complex probe Ca3 (32,34). The Southern blot hybridization patterns of the parent strain and the progeny of each strain were identical (Fig.2B).

FIG. 1. — Pedigrees demonstrating spontaneous in vitro loss of heterozygosity at theMTL locus. Selected progeny of threeMTL-heterozygous parental strains, P37037par, P37039par, and P75063par, that formed opaque-phase sectors were analyzed by PCR amplification ofMTL genes. TheirMTL genotypes are codeda/α forMTL heterozygotes,a forMTLa homozygotes, and α forMTLα homozygotes.

FIG. 2. — Demonstration by PCR of spontaneous in vitro loss of heterozygosity at theMTL locus by strainsP37037 andP37039. (A) PCR analysis of theMTL locus demonstratingMTL heterozygosity of the parent strains [par(a/α)] andMTL homozygosity of the progeny isolate (α/α). TheMTLa1 andMTLα2 genes of each test isolate were amplified by PCR and run in a 1% agarose gel. (B) Southern blot analysis with the complex fingerprinting probe Ca3 confirmed that, in both strains, the heterozygous and homozygous isolates were progeny of the original parental strain.EcoRI-digested DNA was run in a 0.8% agarose gel, Southern blotted, and hybridized with radiolabeled Ca3 fingerprinting probe.

The drug susceptibilities of the three selectedMTL-heterozygous parent strains and a singleMTL-homozygous progeny of each were compared. For strainsP37039 andP75063, there was no significant difference (i.e., more than twofold increase) between the MICs for the parentalMTL heterozygotes and theirMTL-homozygous progeny. For strainP37037, there was no significant difference between theMTL-heterozygous parental isolates andMTL-homozygous progeny for seven of the eight antifungal drugs. In the case of 5-FC, however, theMTL heterozygote was 16-fold less susceptible than theMTL homozygote. Their respective 5-FC MICs were 4.00 and 0.25 μg per ml. Together, these data demonstrate that spontaneousMTL-homozygous progeny that have not been exposed to antifungal drugs are not more resistant to fluconazole or the other tested drugs than theirMTL-heterozygous parents.

Drug susceptibility and the white-opaque transition.

It has been demonstrated that whileMTL-homozygous strains ofC. albicans undergo white-opaque switching,MTL-heterozygous strains do not. Since high-frequency phenotypic switching has been shown to alter drug susceptibility levels (38), the possibility was entertained that the higher proportion of fluconazole-resistant strains in the collection ofMTL-homozygous strains analyzed by Rustad et al. (27) may have been due to expression of the opaque-phase phenotype (9,26,33,36,37). To test this possibility, we compared the susceptibility of white- and opaque-phase cells of sixMTL-homozygous strains to the eight antifungal drugs. For five of the six strains, there was no significant difference between white- and opaque-phase cells in their susceptibility to the eight antifungal drugs. One strain, 19F, exhibited increased resistance to 5-FC in both white- and opaque-phase cells but showed relatively low MICs of the seven other antifungal drugs. In this one strain, white-phase cells were significantly more resistant than opaque-phase cells to 5-FC. Their 5-FC MICs were ≥64.00 and 16.00 μg per ml, respectively. Together, these results indicate that, on average, white- and opaque-phase cells exhibit similar drug susceptibility patterns. Hence, the increase in the proportion of homozygous strains among fluconazole-resistant strains in the collection analyzed by Rustad et al. (27) was not due to increased expression of the opaque-phase phenotype.

DISCUSSION

Although it does not appear that specific drug-resistant strains ofC. albicans have established themselves as significant components of the general colonizing populations ofC. albicans (19,20,30,35), drug-resistant strains have emerged in specific cases of candidiasis. In recent years, a number of genes involved in drug resistance have been identified and cloned (29,39). Among these, genes of the ABC transporter family have been implicated in clinical resistance to the azoles. Two of these genes, CDR3 and CDR4, are regulated by white-opaque switching (2; D. Sanglard, F. Ischer, M. Monod, S. Dogra, R. Prasad, and J. Bille, Abstr. 5th ASM Conf.Candida Candidiasis, abstr. C27, 1999). In addition, a number of other putative drug resistance genes have been recently shown to be differentially regulated by the white-opaque transition (10). The report by Rustad et al. (27) that 22% of fluconazole-resistant strains wereMTL homozygous while only 2% of fluconazole-susceptible strains wereMTL homozygous was interesting in light of the recent discovery that onlyMTL-homozygous strains ofC. albicans undergo white-opaque switching (14,17).

Rustad et al. (27) proposed that the increase in the proportion ofMTL-homozygous strains among fluconazole-resistant strains was either a direct result of becoming homozygous at theMTL locus or homozygous at a gene linked toMTL. To distinguish between these alternatives, we used a strategy opposite that of Rustad et al. (27) in which we tested whether strains selected forMTL homozygosity were more resistant to antifungal drugs. Because homozygosity in natural populations is relatively rare inC. albicans (14), theMTL-homozygous isolates analyzed here were obtained from different geographical locales and different clinical settings. This collection was distinct from the collection analyzed by Rustad et al. (27) in that isolates of their collection were selected solely for fluconazole resistance. If resistance inC. albicans isolates was a direct consequence ofMTL homozygosity, it would be expected that naturally occurringMTL-homozygous isolates that have not been exposed to fluconazole or other antifungal drugs would nevertheless show intrinsic resistance. None of the 17 isolates had been exposed to fluconazole prior to collection, while two were exposed to antifungal drugs other than fluconazole. We found no elevated levels of resistance to fluconazole and very limited resistance to the seven additional drugs among the 17MTL-homozygous isolates. The twoMTL-homozygous isolates that had been exposed to drug therapy prior to collection were not resistant to the drugs they had been exposed to. These results suggest that the relation betweenMTL homozygosity and fluconazole resistance is not direct, and they are consistent with the alternative hypothesis proposed by Rustad et al., namely, that a gene linked toMTL, which becomes homozygous whenMTL becomes homozygous, is responsible for the acquisition of drug resistance (27). An analysis of the region of chromosome 5 harboring theMTL locus in the Stanford genome database, however, failed to reveal a possible gene candidate (i.e., potential drug resistance gene).ERG11, which is the only known gene involved in fluconazole resistance on chromosome 5, does not appear to be a viable candidate since it is not closely linked to theMTL locus and has been found to be heterozygous in someMTL-homozygous strains (27).

To further test whetherMTL homozygosity leads to drug resistance in the absence of drug exposure, we analyzedMTL-homozygous progeny of threeMTL-heterozygous strains that produceMTL-homozygous progeny at high frequency. In no case did we observe fluconazole resistance or general drug resistance in theMTL-homozygous progeny. Finally, we tested the possibility that the increase inMTL-homozygous isolates among drug-resistant isolates was the result of theirMTL-homozygous-associated characteristic of switching from white to opaque (14,17). A comparison of white- and opaque-phase cells (9,26,33,36,37) from sixMTL-homozygous strains revealed no opaque-phase-specific resistance, thus eliminating this possibility as an explanation of the results reported by Rustad et al. (27).

Among the 17MTL-homozygous isolates analyzed, one was resistant to 5-FC and a second one exhibited intermediary resistance to this drug. In addition, significant differences in susceptibility to 5-FC were observed between white- and opaque-phase cells of one strain and between theMTL-heterozygous parent and theMTL-homozygous progeny of another strain. These changes, however, did not support the acquisition of drug resistance as a result of becoming homozygous.

Together, our results demonstrate thatMTL homozygosity in and of itself is not sufficient to confer fluconazole or general drug resistance. In addition,MTL-homozygous progeny formed spontaneously fromMTL-heterozygous strains exhibit the same levels of susceptibility as theirMTL-heterozygous parent strains. These results are consistent with an alternative hypothesis proposed by Rustad et al. (27) that the higher incidence ofMTL-homozygous strains among fluconazole-resistant strains is the result of associated homozygosity of an unidentified gene linked to theMTL locus. These results are also consistent with the possibility thatMTL-homozygous strains develop drug resistance more rapidly thanMTL-heterozygous strains when exposed to antifungal drugs, a hypothesis now under investigation.

Acknowledgments

We are grateful to S. R. Lockhart for valuable suggestions.

This research was supported by NIH grant AI2392 to D.R.S.

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Drug Resistance Is Not Directly Affected by Mating Type Locus Zygosity inCandida albicans

Claude Pujol

Shawn A Messer

Michael Pfaller

David R Soll

Abstract