Vol. 54, No. 3

MICROBIOLOGICAL REVIEWS, Sept. 1990, p. 226-241 0146-0749/90/030226-16$02.00/0 Copyright © 1990, American Society for Microbiology

Genetics of Candida albicans STEWART SCHERER' AND PAUL T. MAGEE2* Department of Microbiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455,1 and Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 551082

INTRODUCTION ................................................. NATURAL HETEROZYGOSITY AND GENETIC DIVERSITY .............................................. GENOME OF C. ALBICANS .................................................

226 227

227

Electrophoretic Karyotype .................................................

227

Cloned Genes .................................................

228

Repeated Sequences .................................................. Molecular Epidemiology and Taxonomy .................................................

230

230

DNA TRANSFORMATION .................................................. Transformation Protocols ................................................. Replication of the Transforming DNA ................................................. Directed Chromosomal Mutations ................................................. PARASEXUAL GENETICS ................................................. Spheroplast Fusion .................................................. Genetic Mapping .................................................. GENETIC INSTABILITY AND PHENOTYPIC VARIATION ................................................. VIRULENCE DETERMINANTS .................................................. DRUG RESISTANCE .................................................

Fluoropyrimidines ................................................. Drugs That Target Sterol Pathways ................................................. CANDIDA SPECIES ................................................. C. stellatoidea .......................................................... Other Medically Important Candida species .................................................. CONCLUSIONS AND PROSPECTS ................................................. ACKNOWLEDGMENTS ................................................. LITERATURE CITED .................................................

231 231

231 231 232 232 233 233 235 236

236 237 237 237 238 238 238 238

morphology to differences in cell shape, surface, and permeability. These two properties may be important in pathogenesis (119) and are of great intrinsic interest as biological processes (109). In the past 10 years, work on the genetics and molecular biology of C. albicans has increased greatly. Beginning with the demonstration that the organism as usually isolated is diploid and naturally heterozygous (80, 141), the modern era of Candida genetics has moved rapidly from the isolation of mutants and the initial use of parasexual genetics to molecular genetics and electrophoretic karyotyping. The result of these advances is that a significant amount is becoming known about the genome and the genetic map of the organism. More importantly, the techniques necessary to gain new knowledge even more rapidly are becoming available. This is in large part a result of the similarity of the organism with Saccharomyces cerevisiae at the molecular level. The techniques used for the molecular and genetic manipulation of C. albicans have been the subject of recent reviews (56, 72). At the same time that the genetics of the organism was becoming more approachable, careful studies of its biology were leading to new information about some of its characteristics and to the reinterpretation of old information. It therefore seems appropriate at this time to review the genetics of C. albicans, both classical and molecular, and the resulting insights into its virulence, its epidemiology, and its relationships with other yeasts.

INTRODUCTION Candida albicans is an imperfect yeast which exists as a commensal with a large number of animals (93, 109). In individuals with impaired immunity or in patients who have suffered an insult to their normal microflora, infections with C. albicans or related species such as Candida tropicalis can be a serious medical problem (21, 65, 79). Life-threatening Candida infections are frequently seen in transplant recipients. In some studies, more than one third of cancer patients at autopsy had evidence of systemic Candida infections. Candida esophagitis is among the most frequent of the opportunistic infections seen in acquired immunodeficiency syndrome patients. Vaginal Candida infections are a significant source of morbidity among women of childbearing age. C. albicans also causes oral thrush in infants. The limited number of effective and safe antifungal antibiotics exacerbates these problems. Given the serious nature of diseases caused by the organism and the role of genetics in our understanding of bacterial pathogenesis, development of the genetics of C. albicans is an important endeavor. C. albicans is capable of a yeast-to-hyphal-phase transition (dimorphic transition) and a variety of high-frequency phenotypic transitions, ranging from differences in colony

*

...

Corresponding author. 226

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NATURAL HETEROZYGOSITY AND GENETIC DIVERSITY

The early studies on the genetics of C. albicans involved mainly the isolation of auxotrophic variants (6, 58). UV irradiation was the mutagen used, and a variety of different phenotypes was isolated. The major rationale for these experiments was to determine whether various phenotypes were correlated with a change in virulence. From a geneticist's point of view, the interest of these studies in that a variety of phenotypes was isolated with some ease and that there was a strong similarity to many of the phenotypes of S. cerevisiae mutants such as red adenine auxotrophs. These results rendered most plausible the hypothesis that the organism was haploid, like the common laboratory strains of S. cerevisiae, and stimulated efforts to find its sexual phase. The question of the ploidy of C. albicans was raised anew by Olaiya and Sogin (80), who carried out DNA measurements on several strains and found that they contained approximately the same amount of DNA as did diploid S. cerevisiae. Furthermore, the survival curves after treatment with UV light, ethyl methanesulfonate, or nitrosoguanidine for C. albicans and diploid S. cerevisiae were very similar. The problems with these experiments are, of course, that one cannot assume similar genome size or DNA repair systems in making interspecies comparisons. In fact, C. albicans apparently lacks a photoreactivation repair system (98). The question was soon resolved by the genetic demonstration that C. albicans is heterozygous at several loci and therefore must be largely diploid. Strong evidence for a diploid genome was put forward by Whelan et al. (141), who showed that for several clinical isolates UV irradiation gave a very biased spectrum of auxotrophs, with methionine requirements predominating. One possible explanation for this was that the strains were heterozygous and that the treatment induced mitotic crossing over, rendering the recessive auxotrophic mutations homozygous. If both progeny of the crossover event survive, one should be able to find a sectored colony with one part homozygous auxotroph and the other homozygous prototroph. This prediction was tested by isolating both the auxotrophic and the prototrophic progeny of the putative crossing-over event and asking whether the prototroph still gave a biased auxotrophic spectrum. In fact, the spectrum was random, as would be expected if the original heterozygosity had been eliminated by mitotic recombination. Furthermore, revertants of the auxotrophic progeny behaved like the original parent, in that they gave the same auxotroph almost exclusively. The simplest explanation for these experiments is that the organism is diploid. Extending this observation, Poulter et al. (87) found that multiply auxotrophic strains could be serially reverted to give prototrophs which, when irradiated, gave back double or triple auxotrophs, indicating that the auxotrophs were produced by mitotic crossing over and that the markers were linked. The extent of natural heterozygosity has been demonstrated in several ways. As mentioned above, Whelan et al. (138, 141) showed that a large number of clinical isolates are heterozygous for recessive mutations which lead to auxotrophies. Poulter (84) estimated that approximately 5% of C. albicans isolates are heterozygous for one of the suf mutations (leading to inability to reduce sulfite) and that an additional 5% are heterozygous for a variety of other mutations. Since these studies used the observation that a limited spectrum of auxotrophs was generated by UV irradiation as the criterion for heterozygosity, the numbers must be re-

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garded as a lower limit. Heterozygosity near the centromere would rarely be revealed in these experiments, and leaky mutations might escape detection. So too, of course, would temperature-sensitive mutations (most of the experiments were done at 34°C) and those which affect a parameter such as growth on a carbon source other than glucose. Among the other types of heterozygosity detectable by growth phenotype are resistance to 5-fluorocytosine (5-FC) (17, 135), failure to elaborate an extracellular protease (15), and recessive lethal mutations (142). One can therefore assume that more than 10% of C. albicans isolates are heterozygous for recessive mutations. The extent of DNA polymorphism and associated natural heterozygosity is described below. The existence of recessive lethal mutations was demonstrated by Whelan and Soll with strain Ca526 (142). Using a suf gene as marker, they demonstrated that the presence or absence of colonies sectored for prototrophy and auxotrophy, expected from a mitotic crossover, could be explained by postulating the existence of recessive lethal mutations linked to the auxotrophy. No other such mutations have been reported, but that may be because it requires a fairly elaborate genetic analysis to show their existence. If they are common, it makes the existence of an independent haploid phase problematic. An important implication of the elevated ploidy of Candida strains is that it provides a significantly greater potential for genetic diversity than would be found in a haploid strain. Presently we know of few organisms which totally lack the possibility of genetic exchange, and our view of C. albicans as an imperfect fungus may well be altered as we find out more about its biology. However, if it has lost its sexual cycle, then diploidy, heterozygosity, mitotic recombination, and phenotypic instability may be its mode of generating and maintaining genetic diversity. GENOME OF C. ALBICANS Many of the earliest studies of the C. albicans genome focused on determination of the ploidy of clinical isolates (90). Direct measurements of DNA content by diphenylamine assays (90) or flow cytometry (19) gave values near 37 fg. These numbers are very close to the value obtained with diploid S. cerevisiae. C. albicans DNA contains about 0.1% 5-methylcytosine during growth in the yeast phase and significantly less during mycelial growth (95). Genome sizes calculated by reassociation kinetics (19.6 and 14.8 fg by different methods) agree well with the DNA-per-cell values if one assumes a diploid genome (90). The range of values given above corresponds to a haploid genome size (including mitochondrial DNA) of 14 to 18 megabases (Mb). Examination of the electrophoretic karyotype revealed on pulsedfield gels has provided graphic evidence of the diploid nature of C. albicans.

Electrophoretic Karyotype C. albicans was one of the first species examined by pulsed-field electrophoresis (66, 71, 78, 116, 117, 132). Its chromosomes were found to be, on average, somewhat larger than those of S. cerevisiae and were useful for testing parameters for the resolution of molecules in the 1- to 3-Mb range (82). Aside from the mitochondrial DNA, no small, circular plasmids have been found in C. albicans. The natural diversity of the species and its diploid nature complicated the determination of the number of distinct chromosomes. The existing genetic data indicated that several

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SCHERER AND MAGEE WO-1 .24

FC18 A

CT1

RDNI

CDC21 ADE2 CHR4

-

A CT SNC'. L YS 2

LYS2

DFRI1 CHR?4

L YS? SNCJ

CHR6 DFR1 CHIR6 SNC:;

FIG. 1. C. albicans karyotypes. The electrophoretic karyotypes and genetic maps of strains WO-1.24 (an auxotrophic derivative of strain WO-1) and reference strain FC18 are compared. WO-1.24 has an additional chromosome with a mobility intermediate between standards 4 and 5 plus a much smaller chromosome. The positions of the bands are relative order on contour-clamped homogeneous electric field gels (132) and do not represent absolute mobility. Cloned genes in the central column label bands of similar size in both strains. Probes listed on the sides highlight differences between the strains. Sources of data are listed in Table 2. Additional data for WO-1.24 are from B. B. Magee (personal communication). For a discussion of the largest band in the karyotype, see the text. The sequence labeled "LYS2" does not complement all S. cerevisiae lys2 mutants (B. B. Magee, personal communication).

linkage groups would be found, but these data were not adequate to provide an estimate for the total number of chromosomes. There is substantial chromosome length polymorphism when different C. albicans strains are compared (66, 70, 71, 78, 116). These polymorphisms may be homozygous or heterozygous. It is the rule, rather than the exception, that in any strain at least one pair of homologous chromosomes can be resolved from each other by pulsed-field gel electrophoresis. Since molecules must differ in size by at least tens of kilobases to be resolved by this method, this result would suggest that extensive regions of material were present on one homolog and absent on the other. Superimposed upon the chromosome length polymorphism is some instability in the karyotype of strains maintained in the laboratory, variation in ploidy, and the presence of additional, much smaller chromosomes in some strains. Suzuki et al. (127) have reported changes in ploidy and chromosome rearrangements associated with changes in morphology. Strain WO-1, the white-opaque switching strain, has an additional, much smaller chromosome consisting at least in part of sequences from the conventional set and also contains a second rearranged chromosome (Fig. 1). Kelly et al. (49) found that one strain, SGY129, appeared to be tetrasomic for the URA3containing chromosome (chromosome 3), with each of two homologs being present twice.

Group

Without an extensive genetic map to serve. as a guide, a molecular approach was taken to analyze the electrophoretic karyotype and determine which bands represent distinct linkage groups. An extensive array of cloned single-copy sequences of both known and unknown functions was used to probe Southern blots of separated chromosome-sized DNA molecules (70). Analysis of these data led to the conclusion that several C. albicans strains have seven pairs of chromosomes. These seven linkage groups would contain more than 13 Mb of DNA. With the addition of the multicopy 41-kb mitochondrial genome, there is reasonably good agreement with the genome size calculated above. A complication in this analysis is the possibility that the chromosomes of two distinct linkage groups would have the same electrophoretic mobility. In strain 1012A, there is clearly an additional large DNA molecule in the electrophoretic karyotype (63). Its mobility is very similar to that of chromosome 1 in strain FC18 (the largest), and it contains the rDNA but lacks ACT] (Table 1). Evidence is accumulating that the largest band in the electrophoretic karyotype of reference strain FC18 may consist of two physical linkage groups more easily resolved in other strains (E. RustchenkoBulgac, personal communication). The sizes of the largest chromosomes are not known with sufficient accuracy to use comparison with the genome size to resolve the issue. These data and the extensive variability in the smaller chromosomes described above lead one to the view that there is no standard karyotype for the species. The physical map derived from a small number of reference strains may, in many instances, be difficult to reconcile with data obtained from a particular clinical isolate of interest. The extensive natural variation in the karyotype makes it very difficult to imagine an efficient meiotic cycle for the species. For the purposes of this review, we will use an existing seven-chromosome numbering system as a reference (numbered 1 through 7, largest to smallest [70]) with the former chromosome 1 designated L for the large chromosomes. At present, there are insufficient data to assign unambiguously the many cloned genes derived from the large chromosomes into two distinct physical linkage groups. These data are summarized in Table 1.

Cloned Genes Most of the cloned C. albicans genes have been isolated through their counterparts in S. cerevisiae. Several approaches have been taken including complementation (27, 42, 52, 54, 57, 94), sequence homology (67, 75, 114), and the ability of certain C. albicans sequences to confer new phenotypes on Saccharomyces strains (70). Genes cloned in this fashion range from the highly conserved actin (75) and tubulin (114) genes to genes involved in amino acid biosynthesis (94) and sugar utilization (70). The first gene isolated

TABLE 1. Assignment of cloned genes to the karyotype of C. albicans FC18' Size (kb)b DNA probe(s)

LC 3,000 ACTI ADEI CDC3 CDCio GAL] MGLI RDN1 SOR9 TRPI TUB2 2 2,500 CDC21 ERG7 HEM3 HIS3 HPTI PRAI RAS] 3 ADE2 ILV2 SOR2 URA3 2,000 4 1,800 L YS1 5 CAGI ERGIJ 1,500 6 1,300 ARSI BEN] 7 1,200 ARG57 DFRI LEU2 a For sources of data, see Tables 2, 3, and 4. b The chromosome sizes are estimates and will vary considerably when different strains are examined. c Group L may consist of two physical linkage groups (see text).

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TABLE 2. Genes isolated through S. cerevisiae function Gene (protein)"

Chromosomeb

Isolation method'

Verification method

ACT] (actin) ADEI (CAIR:aspartate ligase) ADE2 (AIR carboxylase) ARG4 (arginosuccinate lyase) BEN] CAGI (G protein, ot subunit) CDC3 CDC1O CDC21 (thymidylate synthase)e CHSI (chitin synthase I) DFRJ (dihydrofolate reductase) ERG7 (2,3-oxidosqualene cyclase) ERGll (lanosterol-14a-demethylase)f GAL] (galactokinase) HEM3 (uroporphyrin-I synthase) HIS3 (imidazole glycerol phosphate-dehydratase) HPTJ (hypoxanthine:guanine phosphoribosyl transferase) ILV2 (acetolactate pyruvate lyase) LEU2 (isopropylmalate dehydrogenase) MGLI PRAI RAS] SOR2 SOR9 STA1 (glucoamylase) TRPJ (PRA isomerase) TUB2 (,3-tubulin) URA3 (orotidine monophosphate decarboxylase)

L L 3

Homology Complementation Complementation Complementation New function Homology Complementation Complementation Complementation Complementation Homology Complementation Complementation Complementation Complementation Complementation Complementation Homology Complementation New function Homology Homology New function New function New function Complementation Homology Complementation

Sequence Complemeonts C. albicans adel Complemeents C. albicans ade2

6 5 L L 2 7 2 5 L 2 2 2 3 7 L 2 2 3 L

L L 3

Referencesd

75 A 54 B 70 D Sequence C Sequence C Sequence 111 Sequence 56 4, 55 Sequence 50, E 52, 62, E Sequence 70 Phenotypee after gene disruption 57, E 70, 94 A D Function iin S. cerevisiae Phenotypee after gene disruption 42, 48, E 70

67 Function i in

S. cerevisiae

D

70

Sequence Phenotypee after

gene

70 56 94 114 disruption 27, 49, 70

"Gene-enzyme relationships are from Jones and Fink (44). Nomenclature conforms to that used with S. cerevisiae. b Chromosome assignments are as in Table 1. c Isolation methods are sequence homology with a Saccharomyces gene, complementation of existing S. cerevisiae mutants, or the ability of the Candida sequence to confer a new selectable phenotype on S. cerevisiae. d A, S. Scherer, unpublished data; B, P. Russel and S. Wagner, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, F80, p. 471; C, B. DiDomenico, personal communication; D, J. Hicks, personal communication; E, C. Thrash-Bingham and J. Gorman, unpublished data. e CDC21 = TMPI. f ERGII ERG16.

by complementation was URA3, which, like its S. cerevisiae cognate, also functions in Escherichia coli (27). Genes corresponding to the markers in standard Saccharomyces transformation hosts, such as LEU2, HIS3, and TRPJ, were readily isolated. In some instances it has been possible to verify the identity of these genes by their ability to complement characterized C. albicans mutants; however, the number of C. albicans mutants with known biochemical defects remains relatively small. Two genes in this category are the ADEJ and ADE2 loci, whose mutants are red. Some cloned genes have been used to create Candida mutants through gene disruption methods, and the identity of these sequences is confirmed on the basis that these mutants have a predictable phenotype. Examples in this group include LEU2 and URA3 (48, 49). The nucleotide sequences of several cloned genes, such as those encoding actin and tubulin, have been obtained and confirm their identities. Table 2 summarizes existing data on these cloned single-copy DNA sequences. The ability to confer new phenotypes on S. cerevisiae with Candida genes extends the range of those that might be cloned in Saccharomyces strains. Magee et al. used the inability of S. cerevisiae to utilize certain sugars as the basis for gene isolation based on expression (70). Two clones isolated through their ability to confer resistance in S. cerevisiae to the antibiotics benomyl and methotrexate were found to be identical (M. Fling, personal communication). The sequences encoded neither of the targets of those drugs, and their nature remains unknown. Most studies of expression of Candida genes in S. cerevisiae have used high-copy-

number YEp vectors, but it has been demonstrated for URA3 that complementation can be obtained at one copy per haploid genome (27). Genes such as ADEJ and ADE2, which have played important roles in the development of the parasexual genetic system, are the tools for alignment of the physical and genetic maps of C. albicans. Essential to the completion of this task is the isolation of genes corresponding to Candida auxotrophs whose biochemical defect is unknown. This will decrease reliance on anonymous DNA segments and associated restriction fragment length polymorphisms (RFLPs) for chromosome assignment of linked genes. Recently, Goshorn and Scherer (unpublished data) have been able to isolate several Candida biosynthetic genes by complementation in C. albicans. This approach will provide many new selectable markers for DNA transformation of Candida species and makes possible the isolation of genes affecting functions not found in S. cerevisiae. The frequency at which Candida genes complement S. cerevisiae mutants and the nearly complete set of defined biochemical Saccharomyces mutants (44) should, in many cases, permit rapid identification of the encoded enzyme. These additional cloned singlecopy sequences are

listed in Table 3.

The sequenced Candida genes have conformed with what has been learned about the structure of S. cerevisiae genes. In cases when intervening sequences exist, they contain the highly conserved S. cerevisiae splicing recognition sequences (114). There is substantial codon bias in the abundantly expressed genes, as is seen with other fungi. Work to

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TABLE 3. Additional cloned single-copy sequencesa Name

Chromosome

Function

ARG57C ARS1

7 6

Complements Candida mutant ARS

A

CHR4 CHR6 CHR7 LYSId SNC3 SNC9 TEFI TEF2

4 6 7 4 5 5

Unknown Unknown Unknown

70 B 70 A B B 125 125

Referencesb

Complements Candida mutant Unknown

Unknown Translation factor lot Translation factor la

55, B

a Sequences of unknown function were isolated to provide specific DNA probes for chromosomes 4, 6, and 7 in strain FC18 and the small chromosome in strain WO-1 (SNC3 and SNC9). b A, A. Goshorn, personal communication; B, B. B. Magee, personal communication. c The arginine gene complements the auxotrophy in strain STN57 (128). d The LYS] gene corresponds to the S. cerevisiae function (44) as well as the previous Candida mutant designation (36).

date has not provided information on the precise nature of a Candida promoter. Examination of single-copy genes provides an additional measure of heterozygosity in C. albicans. There has been one published report of such RFLPs, those found for the URA3 locus by Kelly et al. (48). In examining two strains which they had used as recipients for a gene disruption experiment, these workers found that the pattern of EcoRI sites was different on the two homologs in each strain. Interestingly, one pattern of sites appeared in both strains, indicating that at least one homolog was similar in the two Candida isolates.

Repeated Sequences Most of the repeated DNA in C. albicans consists of the mitochondrial DNA and the rDNA tandem array (Table 4). The number of copies of the rDNA has been estimated by digestion of chromosomal DNA with a 6-base recognition enzyme that does not cleave the rDNA (e.g., BamHI) and electrophoresis on pulsed-field gels. Two bands are obtained in the 0.5- to 1-Mb range, which most probably derive from the rDNA genes on each homolog (B. B. Magee, personal communication). This would imply about 40 to 80 copies of the 12- to 14-kb repeat unit per haploid genome. The mitochondrial genome has been analyzed in some detail by Wills et al. (143, 144). It is a 41-kb circle containing a large inverted repeat. It is present in lower copy number than is the rDNA. Candida species contain several dispersed, repeated families of DNA sequences (Table 4). These were sought in TABLE 4. Repeated sequences Familya

No. of copies

(distribution) rDNA 40-80 (tandem array) Mitochondrial DNA ca. 30 (mitochondria) 27A ca. 10 (dispersed) Ca3 ca. 10 (dispersed) Ca7 (telomeres)

Repeat size References (kb)

12-14 41 ca. 15

29, 69 142, 143 105 120-122

120-122

The 27A and Ca3 gene families are homologous, but their precise relationship is not yet established. An additional repeated sequence has been identified in C. albicans (16); however, it also may be related to one of the families listed above (see text). a

several laboratories as a route to sensitive probes for typing C. albicans strains. Two approaches were taken for the identification of such sequences. Both involved hybridization of total genomic DNA probes to clones of C. albicans DNA. In such an experiment, increasing copy number in the genome leads to increased signals in the final hybridization experiment. Scherer and Stevens (106) sought species-specific sequences and probed the same clones in parallel with C. tropicalis DNA to eliminate conserved regions from study. Soll et al. (120-122) used mitochondrial DNA and rDNA sequences as probes to specifically exclude those highly repeated' elements. Several classes of sequences emerged from these efforts (Table 3). One of these families appears to be telomeric (Ca7) and produces new DNA polymorphisms at extremely high rates in some strains. The Ca3 and 27A gene families are specific to C. albicans and Candida stellatoidea. The 27A family produces new DNA polymorphisms in the laboratory at low but detectable rates. New polymorphisms are often seen when drug-resistant mutants are selected. These clones were isolated from different C. albicans strains and used to probe genomic DNA from unrelated strains, so th'eir precise relationship remains unclear; however, the 27A family is homologous to the Ca3 family (S. Scherer, unpublished observations). Ca3 and Ca7 were isolated in the same laboratory and are known to be distinct. Because of the choice of restriction enzymes used, the relationship of an additional gene family isolated by Cutler et al. (16) to all of the repeated sequences described above is unknown. A similar analysis of repeated sequences has proven effective with C. tropicalis (122). Molecular Epidemiology and Taxonomy The extensive DNA polymorphism found in C. albicans and other Candida species has led to the rapid development of a variety of DNA-based typing systems for epidemiologic applications. These methods differ greatly in their resolution and are therefore best suited to somewhat different tasks. Unlike biochemical tests (133), DNA-based methods can reveal many distinct types through common methodology rather than a single plus-or-minus result. However, detection of too many types makes the construction of useful subgroupings with potential predictive value more difficult.' One method makes use of the high level of chromosome length polymorphisms and examines strains for karyotypic differences. As the karyotypes of C. albicans and C. tropicalis are reasonably well conserved, this approach also has potential for species typing (71). This method has proven extremely valuable in discriminating between C. albicans and its closest relative, C. stellatoidea (61). A second approach is simple examination of the fluorescence pattern seen after restriction endonuclease cleavage (usually with EcoRI) followed by electrophoresis on a conventional agarose gel (105). Even a modest degree of sequence divergence will result in a strikingly different digestion pattern. From the background pattern of bands one can easily determine which Candida species is present, and the repeated rDNA and mitochondrial DNA add sufficient polymorphism to distinguish dozens of subtypes within C. albicans (76, 124). EcoRI digestion of C. albicans DNA yields three prominent bands derived from the rDNA (69). The middle of these three bands has been found in only two sizes, 3.7 and 4.2 kb. Strains with the 3.7-kb band predominate; those with the the 4.2-kb band include about 10% of recent clinical isolates (124). The other two fragments are highly

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polymorphic. This technique has been used to examine the relationship to one another of C. albicans isolates found in infections of an individual, to study venereal and nosocomial transmission, and to correlate DNA types with the previously described groupings based on biotyping studies (76, 124). Southern blots derived from these gels can be used for subsequent detection of RFLPs. Infections of an individual are generally clonal in origin, and indistinguishable isolates can be recovered from sexual partners and from patients in the same hospital. There is neither strong concordance nor random association of groups derived by molecular methods and the earlier phenotypic groups (124). Several DNA probes have proven useful for RFLP detection in C. albicans. Species can be verified by using the relatively conserved gene encoding actin (1, 75). Strains can be typed by using the repeated but homogeneous mitochondrial DNA (81). The most sensitive typing methods make use of the dispersed, repeated sequences described above (23, 106, 120-122). These probes will easily distinguish all C. albicans strains that are not epidemiologically related and are capable of detecting polymorphism that arises during the course of a single infection. In principle, these probes could be used to determine the lineages of cells in an infection in much the same way that mosaics are used to analyze development of multicellular organisms. The lack of a sexual cycle precludes the use of mating and the production of viable, fertile meiotic products for the definition of Candida species. As a result, traditional typing schemes have relied on arrays of biochemical and growth tests to distinguish Candida species (133). The development of DNA-based typing schemes has led to a reevaluation of the traditional species designations; in most cases these taxonomic distinctions have held up well. However, Candida parapsilosis appears to include three rather distinct groupings, one of which contains at least one strain typed as Candida mauritiana (105). C. albicans and C. stellatoidea are the only Candida species which produce germ tubes in serum. For these two species, the only reliable difference was that C. albicans could utilize sucrose (60). Molecular methods have led to a reexamination of the relationship of C. albicans to C. stellatoidea, which will be described in detail below. DNA TRANSFORMATION The DNA transformation system developed for C. albicans by Kurtz et al. (54) shares many similarities with those used with other fungi, particularly S. cerevisiae. The keys to its development were the availability of genes cloned through their S. cerevisiae cognates and mutants of the corresponding C. albicans genes. Among the few C. albicans mutants with well-defined deficiencies are the two adenine auxotrophs which accumulate a red pigment. The initial experiments, using an ADE2 gene isolated by complementation in S. cerevisiae, achieved a transformation frequency similar to that seen in the first experiments with S. cerevisiae. As the development of the Candida DNA transformation system has, in many ways, paralleled that of the S. cerevisiae system (8), here we will point out the similarities and differences in the behavior of these yeasts.

Transformation Protocols Several very diverse procedures exist for transformation of S. cerevisiae. One closely resembles spheroplast fusion procedures (37). Osmotically stabilized spheroplasts are

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incubated with DNA in the presence of calcium chloride, polyethylene glycol is added, and the cells are plated in a regeneration agar overlay or onto osmotically stabilized plates. Although a somewhat different protocol was originally described (54), a procedure essentially identical to that used with S. cerevisiae will work well with C. albicans. Spheroplast transformation has proven effective with strains of disparate genetic backgrounds. Although some differences in transformation efficiency have been noted, they are not of the magnitude seen with unrelated S. cerevisiae hosts (8). The set of strains available for testing remains limited to those with mutations in certain cloned genes, but is rapidly increasing. Development of dominant markers for the transformation system will permit the transformation of the more highly virulent prototrophic clinical isolates. In contrast, the lithium procedure (41), which works well with a variety of fungi, is largely ineffective with C. albicans. Several other approaches are available for transformation of fungal cells; these include electroporation (134) and direct transfer during conjugation with bacteria (33). The efficacy of these techniques with Candida species remains to be determined. Replication of the Transforming DNA The Saccharomyces transformation system has thus far proven an excellent model for the modes of replication of the transforming DNA. The initial experiments by Kurtz et al. (54) involved integrating vectors. A modest degree of nonhomologous recombination is seen, but it has not interfered with the targeting of linear molecules for gene disruption experiments (49). Sequences were soon identified that were capable of promoting autonomous replication of the transforming DNA (ARS elements) accompanied by greatly enhanced transformation frequencies. Although their behavior is similar to that seen in S. cerevisiae, the ARS-containing plasmids are largely replicated in Candida species as multimers (55). This subtle distinction may complicate determination of the structure of the transforming DNA and its recovery into E. coli. Multimeric replication of autonomously replicating plasmids is seen in other fungal transformation systems (24). Centromeres are genetically defined by segregation patterns in meiosis and cytologically defined through the appearance of metaphase chromosomes. Candida centromere sequences have not yet been described. Whether they have the simple structure seen in S. cerevisiae (35) or the more complex features of Schizosaccharomyces pombe centromeres (31), the lack of a meiotic cycle will complicate the discrimination of centromeres from other sequences that can enhance mitotic stability of ARS plasmids. Centromeric DNA could be distinguished by the fact that dicentric plasmids constructed in vitro are not tolerated in vivo (73). Directed Chromosomal Mutations

Homologous integration of transforming DNA has permitted the development of methods for the construction in vitro of previously unidentified mutants of C. albicans. These molecular approaches make possible the construction of certain multiply marked strains that are not readily produced through parasexual genetics. Gene disruption procedures also avoid additional deleterious mutations that often accompany conventional mutagenesis. These techniques, although generally similar to those used with S. cerevisiae, are complicated by the diploid nature of C. albicans.

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Two methods have been described by Kelly et al. to circumvent this problem. One approach uses mitotic recombination induced with low-dose UV light to make an altered gene homozygous (49). This procedure should be generally applicable to both single-copy and repeated sequences, but requires advance knowledge of the phenotype of the desired homozygote. This approach was used to construct mutations at the URA3 locus. In this case, the cloned URA3 gene was disrupted in vitro by insertion of the ADE2 gene into the URA3 coding sequences. A linear fragment from this construct with homology to the URA3 locus at both ends was used to transform an ade2 auxotroph. As a consequence, the creation of the ura3 mutant resulted in the loss of the other marker in the transformation host. A second method uses cotransformation of a linear altered gene with a selectable ARS-containing plasmid (48). Cotransformation in C. albicans is quite extensive, and it is relatively easy to identify strains carrying an appropriately marked gene from a collection of such transformants. This method can be repeated to mutate the other homolog, but is less well suited to conserved gene families, as the second step might not be easily targeted to the same locus on the other homolog. This type of approach does not result in the loss of the original selectable marker in the host and has been used in the isolation of a leu2 mutant. The availability of multiply marked C. albicans hosts and the corresponding cloned genes permits the use of a different selectable marker to disrupt each homolog with a different gene. This procedure has been used by Kurtz and Marrinan to construct a hem3 mutant of C. albicans (57). This method and the second method described above have the advantage of not requiring advance knowledge of the phenotype of the mutant being constructed, except that it must be viable. PARASEXUAL GENETICS The first step in the development of the parasexual genetic system was the establishment of efficient procedures for the isolation of homozygous mutants. These mutants could arise by one of at least three mechanisms. The simplest is uncov-

ering a preexisting recessive heterozygous mutation by mitotic crossover or gene conversion. The second is mutagenesis of one homolog followed by uncovering of the induced mutation. A third would be mutagenesis so extensive that both copies of the gene are mutated. In fact, no

studies have been done to determine the relative frequencies of these three events, but analogy with other organisms suggests that they would occur with decreasing frequency as listed. Because of the lack of a sexual cycle, the introduction of more than one marker usually involves at least two rounds of mutagenesis. A large variety of auxotrophs have been isolated from C. albicans, but the enzymatic deficiencies of only a few are known. The earliest identification of the biochemical basis of a mutation was the demonstration that red adenine-requiring mutants fell into two complementation groups, one of which showed intragenetic complementation while the other did not (89). The adenine requirement of the first group was relieved by high CO2 concentrations. Based on these considerations, the mutations were called ade2 and adel, respectively, by analogy with S. cerevisiae. An arginine auxotrophy was characterized by biochemical means as a mutation in the ARG4 gene (25), and a chitobiase mutant has been identified (43). Also, a fatty-acid auxotroph has been reported to have a A9-desaturase deficiency (53). As described above, several defined mutations have been pro-

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duced by molecular approaches. Recently, the defect in a pyrimidine auxotroph was shown to be ura3 by complementation with the cloned gene (29). The diploid nature of C. albicans allows one to establish genetic linkage in a single strain without the need for genetic exchange (87). The simultaneous appearance of two or more markers in a single mutagenic treatment (such as exposure to UV light) is evidence for linkage, since they may be expected to have been revealed by a single mitotic crossover. Multiply heterozygous auxotrophs (produced by serial reversion of homozygotes) can be subjected to UV irradiation to induce mitotic crossing over in order to give information about linkage. Two markers which often appear together in survivors can be considered to be linked. However, this simple analysis may be complicated by gene conversion, which may constitute as much as 10% of the recombination events.

Spheroplast Fusion More extensive information about linkage can be obtained from spheroplast fusion. In this technique, spheroplasts of cells are fused with polyethylene glycol and regenerated on selective medium. Stable fusion products are relatively rare, and strong genetic selections are required to distinguish the resulting hybrids (commonly called fusants) from the parental strains. The first report demonstrating the usefulness of this approach was by Sarachek et al. (101). A series of reports from workers in other laboratories, using the technique for complementation analysis and demonstration of linkage, appeared soon thereafter (22, 46, 47, 88). Spheroplast fusion is not a one-to-one process but involves the initial formation of large syncytia, which can apparently either undergo immediate nuclear fusion or become metastable heterokaryons (100, 102). These heterokaryons can then undergo nuclear fusion to become tetraploids, they can reduce their nuclear number and segregate parental types, or they can undergo transfer of a limited amount of genetic material from one nucleus to the other and segregate recombinants which largely resemble one parent. Sarachek and Henderson (99) demonstrated that prototrophs constructed by spheroplast fusion sometimes contained an uneven ratio of auxotrophic to prototrophic alleles. Transfer of less than the full complement of chromosomes was first postulated by Kakar et al. (47) and later studied by Sarachek et al. (99, 103). These observations suggest that spheroplast fusion is not a simple process; more evidence for this view is the observation of elevated recombination in segregants from a fusion experiment (47). In these experiments, one parent was homozygous for tryptophan auxotrophy and heterozygous for a linked lysine requirement, while the other parent was homozygous for prototrophy for these two genes. Trp- Lys- progeny occurred in segregants from the heterokaryons produced. The only way these could have arisen would be via mitotic crossing over on a trp lys chromosome. These experiments cannot determine whether the event occurred during the fusion process or in the growth of the culture being used; however, Candida heterozygotes are normally extremely stable. The colonies which arise from protoplast fusion can be considered to be of two types: heterokaryons, which segregate progeny lacking the selected markers (often including entire parental phenotypes), and tetraploids or near-tetraploids, which are quite stable. Mitotic crossing over can be used to examine the stable fusants and extend the linkage information significantly. The fact that the fusants are tetra-

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ploid would be expected to make homozygous segregants much less frequent than those from diploids; however, for unknown reasons, this does not seem to be a problem. Furthermore, the genome of the fusants can be reduced to a diploid (or near-diploid) state by heat shock. Treating the cells at 50 to 51°C for 1 to 2 min causes random loss of chromosomes (36). An alternative method for reducing the chromosome number in fusants is to use one parent which is heterozygous for the partially dominant 5-FC resistance allele (140). Seventy percent of resistant variants picked from such hybrids have a diminished DNA content compared with the fusant, and about half of these are at or close to the diploid level. Thus, one can generate a complete parasexual cycle in the organism by either of two methods. Genetic Mapping

The major use of spheroplast fusion to establish linkage and gene order has come from the work of Poulter and co-workers (36, 86-89). Using mitotic recombination and heat shock, they have defined three linkage groups. In addition to these three chromosomes, several other linkage groups have been defined by mutations which segregate in heat shock experiments independently of the defined chromosomes and of each other (E. Rikkerink and P. T. Magee, unpublished observations). The availability of several cloned genes which correspond to mapped mutations has allowed the assignment of bands on the electrophoretic karyotype to the genetically defined chromosomes. The genetic map has been derived from experiments with only a few strains, and although particular linkages seem to be widely conserved (86), the variability of the electrophoretic karyotype suggests that considerable difference may exist between strains. The extent of these differences can be determined only with more data. The most interesting comparisons would be between fresh clinical isolates or strains that have especially interesting biological properties, such as elevated virulence or phenotypic instability, and the laboratory strains being used to construct the genetic map. Ideally, one would like to perform parasexual crosses between these types of strains. The requirement for auxotrophic markers in each parent renders such experiments very difficult with nonlaboratory strains. Recently, however, Goshorn and Scherer (29) have succeeded in isolating a strain carrying a dominant mutation for resistance to mycophenolic acid, one of the few antibiotics effective against C. albicans, in a multiply auxotrophic background. Fusion of this strain, 1006, with a prototroph, can be selected on the basis of mycophenolic acid resistance and prototrophy (29). These experiments have cast further light on the nature of the primary fusants, mainly because mycophenolic acid resistance appears dominant in a monokaryon but recessive in a heterokaryon. The evidence for this interesting property is the drug sensitivity of unstable fusion products that segregate parental types. Thus, any fusion products selected with mycophenolic acid have not spent many generations before nuclear fusion, and prolonged time in the heterokaryotic state is not necessary for the production of fusion products. Nevertheless, so-called exceptional progeny, lacking one or more chromosomes from either parent, arise. The data point to a high degree of coincidence for loss of different linkage groups. This occurs only if they are lost from the same parent (A. Goshorn, personal communication). This agrees well with the notion that a partial set of chromosomes would be transferred to an intact nucleus in the exceptions. Transfer of chromosomes

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between nuclei in heterokaryons has been observed in S. cerevisiae when karl mutants were used to block nuclear fusion (18). The exceptional fusion products may be useful for linkage analysis and strain construction in that recessive markers can be recovered without treatment with UV or heat shock. GENETIC INSTABILITY AND PHENOTYPIC VARIATION Phenotypic variation (often called phenotypic instability or switching) is a phenomenon in C. albicans which involves a heritable change in colony morphology, cell shape, or alterations in other properties such as susceptibility to antifungal agents. Most significantly, the ability to undergo phenotypic variation is itself a heritable trait. Typically, strains which undergo a phenotypic transition will generate, at a relatively high frequency, variants which form colonies that are wrinkled, contain raised rays, or have other morphologies. Although phenotypic variation was noted by many investigators, the significance of the phenomenon was not generally recognized until the work of Slutsky et al. from the Soll laboratory (112, 113). These investigations demonstrated the reversibility of the process, obtained data on the frequency of the changes, showed that the range of colony phenotypes was limited for any given switching system, and provided evidence for the role of UV light as a stimulator of the transition. The common characteristics of phenotypic transitions are that they appear at a frequency about 2 orders of magnitude higher than would be expected for ordinary mutation; they are reversible, which rules out mitotic crossing over as a mechanism for the switch; and they can affect a variety of properties, ranging from the frequency of mycelium formation to cell shape and metabolism. Typically, they are signaled by the appearance of colonies of abnormal morphology, at a frequency of 1% or less on a plate (Fig. 2). The cells in these abnormal colonies often differ from their parents in the frequency of mycelium formation (some grow almost exclusively as pseudomycelia) or other morphological characteristics. Phenotypic transitions have been observed in strains isolated from patients with active Candida infections; however, there is no strong evidence for the role of these processes in pathogenesis (119, 120). One colony morphology phenotypic transition was studied genetically by Pomes et al. (84). Thee investigators found that a parent with a smooth colony phenotype would yield rough progeny after UV irradiation. This segregation was by originally attributed to the uncovering of a recessive genedid mitotic crossing over. However, the segregation pattern not fit this interpretation, since both rough and smooth colonies gave rise to the opposite phenotype at high frequencies, and one or the other would have had to be homozygous for the recessive gene and hence stable. The authors later showed that treatment with the mitotic inhibitor benomyl, which causes chromosome loss via nondisjunction in many fungi, would also lead to phenotypic instability in their parent strain. Spheroplast fusion of two rough switching strains independently induced by benomyl but descended from the same parent gave a smooth fusion product, indicating that two complementation groups are represented (26). The authors demonstrated by segregant production that the still present in the fuparental auxotrophic markers wereinduce sants. The fact that benomyl can phenotypic instaappear bility is surprising, since most C. albicans strains resistant to the drug as measured by inhibition of growth.

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.,.....

__.

_......................... M-z:: .

FIG. 2. Switching of colony morphology. Shown are a variety of colony types obtained after mild UV treatment of a transformant of strain SGY269. Both variant full colonies and sectors are seen. For additional colony types, see reference 112. Magnification, x4.5.

These results, however, are interesting in the light of the work of Suzuki et al. (127), who used pulsed-field gel electrophoresis to show that a strain which gave rise to aberrant colonies had very frequent karyotypic rearrangements, although no specific pattern correlated exclusively with a particular phenotypic state. Similar results have been obtained by Rustchenko-Bulgac et al. (96), using the same strain studied by Slutsky et al. (112). A possible effect of benomyl would be nondisjunction leading to loss of a repressing function which normally prevents switching. The observations that at least two examples of phenotypic instability are recessive (see below) give this model some credence. The transition which has been most carefully studied is the white-opaque system (2, 3, 51, 91, 113), which was originally found in strain WO-1 but has since been observed in at least three other isolates. This transition involves a shift from the usual budding yeast shape (white cells) to an elongated, slipper-shaped cell which buds apically and at a characteristic angle in opaque cells (Fig. 3). Opaque cells are more sensitive than white cells to sulfometuron-methyl, an antifungal agent, and to UV irradiation. They express both a hypha-specific surface antigen and an antigen which appears to be unique to opaque cells. Scanning electron micrographs showed that the latter antigen is distributed in a punctate manner on the cell surface (3). Opaque cells undergo the dimorphic transition only when anchored to a substratum (2). This is not observed with white cells or other C. albicans strains. The hyphae arise from the center of the elongated

opaque cells, a position where they rarely bud. Variability in the frequency of hyphal formation among different opaque clones suggests that they may not be genetically identical, possibly as a consequence of switching. The extensive differences between the karyotype of WO-1 and those of other strains further suggest a connection between chromosome rearrangements and switching. Because white cells grow faster than opaque cells, the frequency of the transition cannot be measured by counting the number of opaque or white colonies in a given population. However, a Luria-Delbruck fluctuation experiment can be used to give an accurate determination of the transition frequency (91). The opaque-to-white transition occurs at about 5 x 10-4 per cell division at room temperature in strain WO-1; the white-to-opaque transition takes place at a slightly lower frequency. At higher temperatures, the whiteto-opaque shift predominates. At 34°C all opaque cells give rise to white colonies. This shift is programmed by as little as 6 to 8 h of exposure to the higher temperature (91). At least two phenotypic transitions have been shown by spheroplast fusion experiments to be recessive to the stable state. Goshorn and Scherer (29) have shown that fusions of strain 655, a wrinkled-colony-producing strain which gives rise to a variety of morphologies, with a stable smooth strain 1006 (see above) leads to nonwrinkled fusants. Interestingly, many of the fusants had phenotypes distinguishable from that of either parent. Similar experiments have shown that fusants of WO-1 (and auxotrophic derivatives of it) with stable strains are stable. Furthermore, heat-shock-induced

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be undergoing endomitosis, while some tetraploids showed nuclear structures similar to those of meiosis II in S. cerevisiae. Therefore, the culture seemed to be in a dynamic state with regard to ploidy. The fact that the tetraploid nature of the cells was reversible made it impossible to test the hypothesis by genetic means. The relationship of the ploidy shift reported by Suzuki et al. (127) to phenotypic instability is unclear, but the authors draw some analogies between the ploidy shift and the chromosomal rearrangements in switching cells.

FIG. 3. Opaque cells of C. albicans. Note the characteristic budding angle, as well as the size and shape of the mother cell. Opaque cells

are

typically 15 to 20 ,um long. Magnification, x3,500.

chromosome loss, presumably leading to loss of the chromosome or chromosomes encoding a repressor, can lead to regaining of the white-opaque transition. Apparent linkage of the switching phenotype with the URA3 gene has suggested that one or more of the genes responsible for the switch in WO-1 lie on chromosome 3. These experiments also exclude the repressor function from chromosome 3, demonstrating that at least two gene products are involved in the control of switching (W. Chu, personal communication). It appears that it will be possible to analyze switching genetically, but the complicated nature of the system and of Candida genetics will make this task challenging. For example, the fact that switching is recessive will complicate attempts to clone genes involved in the transition, since recipient strains will have to be specially constructed to lack the repression function. The demonstration that at least two genes are involved in the colony morphology switching studied by Nombela and co-workers (26, 84) implies that the system may be quite complicated. Finally, it is important to remember that there are potentially two genetic systems involved in any instance of phenotypic instability: the genes involved in the transition, and the genes involved in producing the alternate phenotype. The genes responsible for the switching mechanism may be common to many or all of the phenotypic transitions, and it is only the target genes that vary.

There are two reports about variation in C. albicans ploidy. Suzuki et al. (126, 128) found that one clinical isolate contained both diploid and tetraploid cells. Electron microscopy of the culture showed that some diploid cells seemed to

VIRULENCE DETERMINANTS Unlike bacteria, pathogenic fungi have not in general been shown to have specific virulence factors.The strongest data exist for the capsular polysaccharide of Cryptococcus neoformans (93). Although C. albicans isolates vary significantly in virulence in animal models, there are relatively few genetic data on individual virulence determinants. Auxotrophs have been shown to have reduced virulence (74, 108), and a fusion product of complementing auxotrophs was virulent as a tetraploid (40). However, these biosynthetic pathways cannot be considered virulence factors per se, because almost any mutant with reduced growth rates in vivo might be expected to also have a reduction in virulence. It is also important to show that the presence of the virulence determinants correlates with pathogenic species or strains. Odds has suggested that the major candidates for virulence factors are the ability to form hyphae (also called the yeast-to-hyphal-phase dimorphic transition), the ability to resist phagocytosis, the ability to adhere to epithelial cell surfaces, and the ability to secrete an acid proteinase (79). More recently, an iC3b receptor has been proposed as a C. albicans virulence factor (28). To these one should add the ability to grow well at 37°C. One very effective way to test the role of any of these factors in pathogenicity is to generate two isogenic strains, one lacking the factor via mutation and the other normal, and to compare them in an animal model. Such experiments have been extremely difficult with Candida species in the past because of the obligate diploid nature of the organism. Loss of a particular factor would presumably be due to a recessive mutation, and mitotic recombination is the most likely way that recessive phenotypes appear. However, the product of a mitotic crossover differs from its parent not only by the homozygosity of the mutation, but also by the elimination of any heterozygosity which is centromere distal to the crossover point. This problem can be circumvented by generating the homozygote for the recessive gene and then picking a revertant, making the assumption that only one copy of the gene in question has changed and that no other information has been altered. The parent, the mutant, and the revertant are then tested for virulence. Only a few genetic experiments have been carried out on potential virulence factors in Candida species. These have focused on three of the factors mentioned above: the ability to form true hyphae, adherence, and the secretion of the proteinase. The experiments dealing with the dimorphic transition have usually involved isolation of a variant which is blocked in the formation of either hyphae or yeast cells (13, 39, 108) and testing its virulence compared with that of the parent organism. One set of such experiments indicated that an amycelial variant was more pathogenic than a strain (hOG301) unable to make yeast cells, although both were able to kill mice (108). However, the two strains were derived from different parents, thus vitiating the conclusion.

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ATCC 10261, the parent of hOG301, was more virulent than the mutant. In a later series of experiments from another laboratory, hOG301 was found to be avirulent (39). The difference may be due to a variation in the size of the inoculum used. It is also important to remember that Candida infections are normally seen in hosts with some type of compromise, whereas the experimental infections are generally done with large inocula (105 or more cells per animal) in healthy animals. Interpretation of these experimental infections with regard to human infections requires great caution. In any case, the general conclusion that this particular mutation led to reduced virulence seems clear. Buckley and co-workers isolated a variant which could no longer form germ tubes (118); this strain was much less virulent than its parent, and reversion for germ tube formation restored the virulence (9, 118). A possible interpretation of all these experiments is that both the yeast and the hyphal forms are necessary for efficient pathogenesis in mice. Additional experiments are needed to explore the role of the dimorphic transition in virulence. There have been two genetic tests of the role of adhesion in virulence (10, 64). Calderone et al. (10) showed that cirulenin-resistant mutants with reduced adherence in vitro are also specifically reduced in virulence in an endocarditis model. Their mutants have altered mannoproteins (11, 104). Lehrer and his colleagues, using the same mutants in a mouse vaginitis model, found that virulence was reduced in proportion to the reduction in adherence (64). However, cirulenin-resistant mutants can have pleiotropic properties (38), and not all are reduced in adherence. These studies had the advantage of working with spontaneous mutants unlikely to have additional genetic defects; a difficulty is the lack of a strong selection to obtain revertants for further study. Most C. albicans strains secrete an aspartyl proteinase with a very low pH optimum. Because levels of this enzyme seem to be elevated in virulent strains and because antibodies against it are found in many candidiasis patients, it was proposed to be a virulence factor (123). Macdonald and Odds (68) were the first to find that a strain making reduced levels of the enzyme was reduced in virulence. They showed that the variant was more easily phagocytized than the parent as well. Because there were several differences between the proteinase-deficient variant and the parent, a second group of workers repeated the experiments with a new set of strains (59). They showed that another proteinase-deficient mutant was reduced in virulence compared with the parent, whereas a reversion of the mutation led to regained virulence along with the capacity to make the enzyme. This experiment ruled out the possibility that some of the other changes found in the mutant, such as a reduced growth rate at 37°C, were responsible for the loss of virulence. Recently, one strain that generated proteinase-negative mutants was shown to be heterozygous for the prt mutation by the frequency with which it generated Prt- derivatives both spontaneously and after UV irradiation. For reasons that are unclear, the negative mutants reverted with a very high frequency even under nonselective conditions (15). This may indicate that the mutations were not in the proteinase gene per se, but in some gene which affected functions in addition to the proteinase. Proteinase mutants are often screened by their failure to grow with protein as the sole nitrogen source. As a result, some avirulent, "proteinase" mutants may have other defects (20) such as the inability to transport peptides. Recently, Lott et al. have reported isolation of a C. albicans gene homologous to an S. cerevisiae aspartyl proteinase used in that species for a different function (67). Sequence

analysis of the Candida protein and disruption of the cloned gene should easily determine whether the secreted proteinase has been cloned; however, if the mutants that have been studied prove to lie elsewhere in the genome, the role of the proteinase in Candida virulence will become less certain. The proteinase therefore seems to be the best candidate for a virulence factor so far identified. Pathogenesis is a very complex process, and a large number of functions are no doubt involved. The genetic approach has proven to be the most efficient way to establish the role of individual virulence functions in other organisms. The same will no doubt be true in C. albicans, but the complicated nature of the genetic system requires that the experiments be carefully conceived and very well controlled. DRUG RESISTANCE C. albicans is naturally resistant to a great variety of antibiotics, severely limiting the set available for use against infections (93, 109). A number of additional antibiotics, not suitable for use against infection, have proven useful in the laboratory. As described above, C. albicans strains are quite sensitive to mycophenolic acid, and dominant resistant mutants can be obtained. Recessive nalidixic acid-resistant mutants have been described by Haught and Sarachek (32). Cerulenin-resistant mutants have been characterized bio-

chemically (11, 104), but little is known about their genetics. In this section, the genetics of resistance to drugs in clinical use will be considered.

Fluoropyrimidines The mechanism of action of the compounds in this group depends on their conversion to 5-fluoro-UMP. This molecule is an inhibitor of thymidylate synthetase and has additional toxic effects after its incorporation into nucleic acids. Among the clinically useful compounds, most is known about the genetics of resistance to 5-FC. Recessive mutants have been characterized extensively at the UMP-pyrophosphorylase and cytosine deaminase loci by Whelan et al. (136, 139). Candida species are also sensitive to 5-fluorouracil and 5-fluorouridine, and these drugs have been used to elucidate the biochemical defects in 5-FC-resistant mutants. Whelan et al. demonstrated extensive natural heterozygosity for 5-FC resistance at a locus controlling UMPpyrophosphorylase activity. Heterozygotes were distinguishable from homozygotes for either resistance or sensitivity by their colony size on agar containing 50 ,ug of 5-FC per ml (136). They found that of 137 clinical isolates, 78 (57%) were homozygous for susceptibility, 51 (37%) were heterozygous for resistance, and 8 (6%) were homozygous for resistance (17). It is important to note that no data are given about the history of these strains, and some or most may have been exposed to the drug during treatment of the patient from whom they were isolated. Thus, this heterozygosity may have been selected and the rate found may not be the natural rate of occurrence. S. cerevisiae mutants with dominant 5-FC-resistant mutations at several loci have been found (45), and it seemed likely that such mutants would be obtained in a diploid

organism. Recently, such C. albicans mutants have been recovered (26, 29), but their biochemical defects have not yet been determined. In combination with appropriate recessive markers, these mutants can be used for selection of fusion products with prototrophs, but the frequency of 5-FC-resistant strains (and those that can mutate easily to

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resistance) may make this approach less generally applicable than the use of the mycophenolic acid-resistant mutants described above. C. albicans exhibits some sensitivity to 5-fluoro-orotic acid, and ura3 mutants are resistant to this drug. As a result, it should be possible to select for high-frequency loss of URA3 function, a technique that has proven quite useful with S. cerevisiae (7).

HJSi-

CANDIDA SPECIES

Because of its importance as a common human pathogen, C. albicans has received more attention than several of the other Candida species. Several Candida species are grown on a large scale for the production of specific enzymes or as a source of feed protein. Among the most widely studied are Candida maltosa and Candida utilis. DNA probes have been used to document a rare case of fungemia due to C. utilis (1). The alkane-utilizing C. maltosa has been the subject of more extensive molecular analyses (107). An examination of the 5S RNA sequences of various Candida species indicates that they may be no more similar to C. albicans than is S. cerevisiae. This is not surprising, as the genus Candida is

ACTI CDC21 ADE2 CHR4 LYS2

ADEI SOR9 HIS.:

CHIGC -

Drugs That Target Sterol Pathways Nystatin and amphotericin B both bind to ergosterol in fungal cell membranes and make treated cells permeable to small molecules (34). Despite problems with solubility and toxicity, these agents are widely used for treatment of surface and systemic infections, respectively (93). Resistance to these antibiotics in clinical strains is' extremely infrequent. Studies of resistance to polyene antibiotics by S. cerevisiae indicate that the major mechanism of resistance is mutation in certain steps in the pathway from lanosterol to ergosterol, resulting in the incorporation of precursor sterols in the cell membranes. In S. cerevisiae, the sterol composition of the inner mitochondrial membrane appears to be important for proper function of that organelle (34). C. albicans mutants that accumulate 14-methyl sterols are defective in the formation of hyphae (110). These factors may combine to select against mutants resistant to the polyene antibiotics. The studies to date indicate that the situation is quite similar in C. albicans (83). Mutants with a similar pattern of accumulated sterols generally fail to complement, and only recessive mutants have been isolated. Among the most effective antifungal agents is a group of compounds that target the cytochrome P450 system, which is required for demethylation steps on the pathway from lanosterol to ergosterol. These include the drugs miconazole, ketaconazole, fluconazole, itraconazole, and the as yet unnamed SCH39304 (30, 77, 115, 131). Here the situation in C. albicans appears to be somewhat different from that in S. cerevisiae. There is evidence that Candida nonheme, cytochrome P450 mutants are different from their Saccharomyces counterparts (5). Studies of mutants with mutations in the heme pathway also reveal differences. Sequences capable of complementing S. cerevisiae hem3 mutations have been cloned from C. albicans and used to disrupt the gene in C. albicans. Again, the phenotype was not the same as that of the corresponding Saccharomyces mutant (57). These results underscore the point that although general features of Candida molecular biology may be discerned from studies of model systems such as S. cerevisiae, certain critical features of the biology will have to be developed by genetic analysis of Candida species themselves.

C. albicans

C. stellatoidea

A DE1

237

DFJ? 1

ADE2 LYS2 SR9 CHR6 DFRI

FIG. 4. Karyotypes of C. albicans and C. stellatoidea. The positions of the bands are the relative order on contour-clamped homogeneous electric field gels, and the distances shown are not proportional to mobility. Cloned genes in the central column label bands of similar size in both species. Probes listed on the sides highlight differences between the species. For example, the assignments of ADEI and HIS3 are reversed. C. stellatoidea has five additional smaller chromosomes. Note that the probes for these chromosomes also label sequences on the standard sized set. The three largest of the five extra chromosomes are very similar in mobility on gels. No probe has been found for one of the small chromosomes. Its existence is inferred from a combination of physical and genetic experiments (92). For a discussion of the largest band in the karyotype, see the text.

something of a taxonomic trash bin for asexual budding yeasts. Several pathogenic fungi, previously thought to be imperfect, have been reclassified when their sexual cycles were determined. In this section we would like to draw attention to other members of the Candida group, focusing on those of medical importance. C. stellatoidea The close relationship between C. albicans and C. stellatoidea has been examined by Kwon-Chung et al. in great detail at the molecular level (60, 61). Strains typed as C. stellatoidea fall into two groups: type II appears to be simply Suc- variants of C. albicans, whereas type I has more extensive differences with C. albicans. Although the genomes of C. albicans and type I C. stellatoidea are conserved to the level of individual restriction sites, type I strains have an extensively rearranged karyotype and several additional smaller chromosomes when compared with the typical C. albicans set (Fig. 4). The assignment of C. albicans DNA probes onto the C. stellatoidea electrophoretic karyotype by Rikkerink et al. (92) has revealed the complexity of the rearrangements as well as extensive conservation of linkage relationships. The smaller chromosomes may be simple fragments of the larger ones or may be assembled from several fragments. The sucrose deficiencies in the two groups complement, whereas those tested within type I fail to complement. This homogeneity of type I C. stellatoidea isolates extends to RFLPs seen when using mitochondrial DNA and Ca3 probes. Type I C. stellatoidea shares antigenic determinants with C. albicans serotype B, whereas the type II strains appear to be serotype A. Clearly, two distinct Suc- groups have separated from C. albicans in the recent past. Type I C. stellatoidea strains differ from C. albicans in several quantifiable phenotypes (60). They are more sensi-

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tive to UV light and cycloheximide, have reduced rates of in rich medium, and are less virulent in a mouse model. In addition, they have the ability to produce the secreted proteinase at lower pH than C. albicans and have a highly wrinkled colony morphology reminiscent of those produced by some C. albicans strains. C. stellatoidea is rarely isolated from current patient populations, and existing strains are almost all vaginal isolates. This species might be considered either a predecessor now being outcompeted by the more virulent C. albicans or a highly specialized derivative adapted to growth in the vagina.

growth

Other Medically Important Candida Species Many Candida species are known to cause human infections. Among those incapable of germ tube formation, C. parapsilosis and C. tropicalis are frequently encountered. Additional molecular and genetic analysis is required to determine the precise taxonomic relationships of these imperfect fungi to C. albicans. Sexual cycles have not been determined for these pathogenic Candida species. The DNA-based typing schemes described above for C. albicans have been applied to both of these fungal pathogens. Species-specific DNA probes have been developed for C. tropicalis (122), and mitochondrial DNA polymorphisms have been used to examine C. parapsilosis (12). DNA fingerprinting methods are readily applied to all of the Candida species. C. parapsilosis and C. tropicalis have been the subjects of both molecular and genetic investigation. Genetic experiments suggest that both species are diploid. Natural heterozygosity has been found in C. parapsilosis (137). C. tropicalis has been the subject of more extensive investigations, in part due to interest in examination of peroxisome biogenesis. Several genes have been cloned from C. tropicalis, including those for peroxisomal enzymes (130) and the alkane-inducible cytochrome P450 (97). One approach to advancing the genetics of these Candida species is the use of interspecific parasexual crosses (129). Corner and Poulter (14) have used interspecific complementation with C. albicans adenine auxotrophs to assign defects in mutants and have developed enrichment procedures for auxotrophs by using inositolless death. It has also been possible to use C. albicans ARS-containing cloning vectors for transformation of C. tropicalis adel and ade2 mutants (D. Sanglard, personal communication).

CONCLUSIONS AND PROSPECTS In the past several years, the molecular genetics of Candida species has undergone several major developments. The parasexual genetic system has developed to a point at which it can be effectively applied to biological questions. Physical mapping methods are permitting rapid creation of a genetic map for C. albicans. The development of a DNA transformation system and allied procedures for chromosomal mutagenesis has allowed the application of many of the techniques previously available only in the major fungal genetic systems to one of the most significant fungal pathogens. Molecular methods have made great progress in clarifying both the epidemiology and taxonomy of Candida species. One point not discussed above in connection with the C. albicans genetic system is a wild-type or reference strain. Individual laboratories have chosen a variety of standard strains or recent clinical isolates suitable for the problems they were addressing. Further development of C. albicans

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genetics will require increased integration of these data. Several characteristics would be desirable in such a reference strain. On the molecular level, high-frequency DNA transformation and a karyotype which resolves the linkage groups would be desirable. Aneuploidy or unusual levels of heterozygosity would greatly complicate genetic analysis. A multiply marked reference strain would facilitate genetics, as mapping procedures for C. albicans are far more efficient with the markers in cis; however, such a strain would not be likely to exhibit virulence in animals. Clearly, this choice is not a simple one and is complicated by the recent observation that fundamental properties such as linkage relationships can show intraspecific variation. Interest in the Candida system is driven in large part by its importance as a human pathogen. A by-product of this work has been extensive examination of the diversity within the species and its significance. In attempting to learn how Candida species have adapted to life in a mammalian host and evolved methods to evade or defeat the host defenses, much new knowledge will be gained about more general questions of evolution at the molecular level. ACKNOWLEDGMENTS We thank W. Chu, B. DiDomenico, M. Fling, J. Gorman, A. Goshom, J. Hicks, R. Kelley, J. Kwon-Chung, B. B. Magee, E.

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Genetics of Candida albicans.

Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in indi...
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