Search

Candida albicans is a diploid organism that exhibits high levels of heterozygosity. Although the precise manner by which this heterozygosity provides advantage for the commensal/pathogenic life styles of C. albicans is not known, heterozygous markers are themselves useful for studying genomic rearrangements, which occur frequently in C. albicans. Treatment of CAI-4 with UV light yielded histidine auxotrophs which could be complemented by HIS4, suggesting that strain CAI-4 is heterozygous for HIS4. These auxotrophs appeared to have undergone mitotic recombination and/or chromosome loss. As expected from a heterozygote, disruption of the functional allele of HIS4 resulted in a his4::hisG-URA3-hisG strain that is auxotrophic for histidine. Sequencing of random clones of the HIS4 ORF from CAI-4 and its precursor SC5314 revealed the presence of 11 SNPs, seven synonymous and four non-synonymous. Site-directed mutagenesis indicates that only one of those SNPs, T929G (Gly310Val), is responsible for the non-functionality of the encoded enzyme. HIS4 analysis of five commonly used laboratory strains is reported. This study provides a new, easily measured nutritional marker that can be used in future genetic studies in C. albicans.

The yeast Candida albicans, a commensal colonizer and occasional pathogen of humans, has a rudimentary mating ability. However, mating is a cumbersome process that has never been observed outside the laboratory, and the population structure of the species is predominantly clonal. Here we discuss recent findings that indicate that mating ability is under selection in C. albicans, i.e. that it is a biologically relevant process. C. albicans strains can only mate after they have sustained genetic damage. We propose that the rescue of such damaged strains by mating may be the primary reason why mating ability is under selection.

The yeast Candida albicans can mate. However, in the natural environment mating may generate progeny (fusants) fitter than clonal lineages too rarely to render mating biologically significant: C. albicans has never been observed to mate in its natural environment, the human host, and the population structure of the species is largely clonal. It seems incapable of meiosis, and most isolates are diploid and carry both mating-type-like (MTL) locus alleles, preventing mating. Only chromosome loss or localized loss of heterozygosity can generate mating-competent cells, and recombination of parental alleles is limited. To determine if mating is a biologically significant process, we investigated if mating is under selection. The ratio of nonsynonymous to synonymous mutations in mating genes and the frequency of mutations abolishing mating indicated that mating is under selection. The MTL locus is located on chromosome 5, and when we induced chromosome 5 loss in 10 clinical isolates, most of the resulting MTL-homozygotes could mate with each other, producing fusants. In laboratory culture, a novel environment favoring novel genotypes, some fusants grew faster than their parents, in which loss of heterozygosity had reduced growth rates, and also faster than their MTL-heterozygous ancestors—albeit often only after serial propagation. In a small number of experiments in which co-inoculation of an oral colonization model with MTL-homozygotes yielded small numbers of fusants, their numbers declined over time relative to those of the parents. Overall, our results indicate that mating generates genotypes superior to existing MTL-heterozygotes often enough to be under selection.

Candida albicans is the most common fungal pathogen of humans. The recent discovery of sexuality in this organism has led to the demonstration of a mating type locus which is usually heterozygous, although some isolates are homozygous. Tetraploids can be formed between homozygotes of the opposite mating type. However, the role of the mating process and tetraploid formation in virulence has not been investigated. We describe here experiments using a murine model of disseminated candidiasis which demonstrate that in three strains, including CAI-4, the most commonly used strain background, tetraploids are less virulent than diploids and can undergo changes in ploidy during infection. In contrast to reports with other strains, we find that MTL homozygotes are almost as virulent as the heterozygotes. These results show that the level of ploidy in Candida albicans can affect virulence, but the mating type configuration does not necessarily do so.

The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans.

Most Candida albicans strains are heterozygous at the MTL (mating-type-like) locus, but mating occurs in hemi- or homozygous strains. The white-opaque switch process is repressed by the heterodimer of the MTLa1 and MTLalpha2 gene products, while mating genes are induced by a2 and alpha1. Mating occurs in opaque cells and produces tetraploid progeny. A small percentage (3-7%) of clinical isolates are homozygous at the MTL locus and most are mating-competent. MTL gene expression is controlled in part by a gene which activates MTLalpha genes and represses MTLa genes in response to hemoglobin. A failure to find meiosis and the lack of evidence of mating in vivo, together with some of the properties of opaque cells, leads to the suggestion that mating may have persisted because the tightly associated switch facilitates the commensal lifestyle of this fungus.

Summary One hundred and twenty Candida albicans clinical isolates from the late 1980s and early 1990s were examined for homozygosity at the MTL locus. Of these, 108 were heterozygous (MTLa/MTLα), whereas seven were MTLa and five were MTLα. Five of the homozygous isolates were able to switch to the opaque cell morphology, while opaque cells were not detectable among the remaining seven. Nevertheless, all but one of the isolates homozygous at the MTL locus were shown to mate and to yield cells containing markers from both parents; the non-mater was found to have a frameshift in the MTLα1 gene. In contrast to Saccharomyces cerevisiae, C. albicans homozygotes with no active MTL allele failed to mate rather than mating as a cells. There was no correlation between homozygosity and fluconazole resistance, mating and fluconazole resistance or switching and fluconazole resistance, in part because most of the strains were isolated before the widespread use of this antifungal agent, and only three were in fact drug resistant. Ten of the 12 homozygotes had rearranged karyotypes involving one or more homologue of chromosomes 4, 5, 6 and 7. We suggest that karyotypic rearrangement, drug resistance and homozygosity come about as the result of induction of hyper-recombination during the infection process; hence, they tend to occur together, but each is the independent result of the same event. Furthermore, as clinical strains can mate and form tetraploids, mating and marker exchange are likely to be a significant part of the life cycle of C. albicans in vivo.

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura + derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.

Variation of the electrophoretic karyotype is common among clinical strains of Candida albicans and chromosome translocation is considered one of the causes of karyotypic variation. Such chromosome translocations may be a mechanism to confer phenotypic diversity on the imperfect fungus C. albicans. A clinical strain, TCH23, from a vaginal candidiasis patient shows distinct carbohydrate assimilation profile, serotype B, no chlamydospore formation and an atypical karyotype (Asakura et al., 1991). To examine the taxonomic relationship among C. albicans, Candida dubliniensis and this strain, we sequenced the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA. The ITS1 sequence of TCH23 was identical with that of C. albicans but not of C. dubliniensis. Thus, strain TCH23 was classified as a variant of C. albicans with an atypical phenotype. The chromosomal DNAs of this strain were resolved into 13 bands on pulse-field gel electrophoresis (PFGE). Using DNA probes located at or near both ends of each chromosome of C. albicans, we investigated the chromosome organization of this strain. Referring to the SfiI map of C. albicans 1006 (Chu et al., 1993), we found that seven chromosomal DNA bands in strain TCH23 were reciprocal chromosome translocations. One homologue from chromosomes 1, 2 and 6 and both homologues from chromosomes 4 and 7 participated in these events. One translocation product was composed of three SfiI fragments, one each from chromosomes 2, 4 and 7. We deduced the breakpoints of chromosome translocation from the physical map of this strain; between 1J and 1J1, between 2A and 2U, both ends of 4F2, between 6C and 6O and both ends of 7F. Copyright © 2001 John Wiley & Sons, Ltd.

Although the diploid fungus Candida albicans , a human pathogen, has been thought to have no sexual cycle, it normally possesses mating-type–like orthologs ( MTL ) of both of the Saccharomyces cerevisiae mating-type genes ( MAT ) a and α. When strains containing only MTL a or MTL α were constructed by the loss of one homolog of chromosome 5, the site of the MTL loci, MTL a and MTL α strains mated, but like mating types did not. Evidence for mating included formation of stable prototrophs from strains with complementing auxotrophic markers; these contained both MTL alleles and molecular markers from both parents and were tetraploid in DNA content and mononucleate.

In Saccharomyces cerevisiae MATa cells, export of the a-factor mating pheromone is mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of transporters and a close homologue of mammalian multidrug transporter P-glycoproteins (Pgps). We have used functional complementation of a ste6delta mutation to isolate a gene encoding an ABC transporter capable of a-factor export from the pathogenic yeast, Candida albicans. This gene codes for a 1323-amino acid protein with an intramolecular duplicated structure, each repeated half containing six potential hydrophobic transmembrane segments and a hydrophilic domain with consensus sequences for an ATP-binding fold. The predicted protein displays significant sequence similarity to S. cerevisiae Ste6p and mammalian Pgps. The gene has been named HST6, for homologue of STE6. A high degree of structural conservation between the STE6 and the HST6 loci with respect to DNA sequence, physical linkage and transcriptional arrangement indicates that HST6 is the C. albicans orthologue of the S. cerevisiae STE6 gene. We show that the HST6 gene is transcribed in a haploid-specific manner in S. cerevisiae, consistent with the presence in its promoter of a consensus sequence for Mata1p-Matalpha2p binding known to mediate the repression of haploid-specific genes in S. cerevisiae diploid cells. In C. albicans, HST6 is expressed constitutively at high levels in the different cell types analysed (yeast, hyphae, white and opaque), demonstrating that HST6 transcription is not repressed in this diploid yeast, unlike in diploid S. cerevisiae, and suggesting a basic biological function for the Hst6p transporter in C. albicans. The strong similarity between Hst6p and the multidrug transporter Pgps also raises the possibility that Hst6p could be involved in resistance to antifungal drugs in C. albicans.

The DNA sequence of a Candida albicans genomic fragment known to complement the arginine mutation designated arg57 in strain 1006 contains an ORF of 1404 nucleotides (nt) predicting a protein of 468 amino acids (aa). Database searches indicated that the deduced protein shares 75% identity and 85% similarity with the ARG4 protein of Saccharomyces cerevisiae. Analysis of the percent aa identity between C. albicans and S. cerevisiae sequences included in available databases suggested these values are within the range expected for biosynthetic enzymes from the two organisms which share similar function. Experiments to isolate C. albicans ARG4 by complementation in an arg4 strain of S. cerevisiae yielded a plasmid (pARG4-1) with a restriction map identical to that of the sequenced clone. From these data, we conclude that the gene previously designated ARG57 is in fact ARG4 encoding the enzyme argininosuccinate lyase (ASL). These results were unexpected, since ARG57 had been localized to chromosome 7, while a mutation causing an ASL deficiency had been linked to ade1, which is on chromosome R. Transformation of C. albicans strains with pARG4-1 indicated it complemented the arginine auxotrophy in strains TMSU221 and 1435, a derivative of 1006. Examination of commonly utilized C. albicans arginine auxotrophs by spheroplast fusion analysis indicated these strains comprise two complementation groups: one consisting of 1006 and TMSU221, which are arg4, and the other of A642, hOG318, hOG357, FC18-6 and WC-5-4, which possess an undefined defect in the arginine biosynthetic pathway which we designate arg100.

Analysis of the karyotypes of multiple Candida albicans isolates by pulsed-field electrophoresis confirms the observation by Lasker et al. of eight chromosomes. The genes previously assigned to chromosome 1 in fact fall into two groups, one (including ADE1, SOR9, and CDC10) is linked to the ribosomal DNA genes on a chromosome called R, whereas the others are found on chromosome 1. Chromosome R varies in electrophoretic mobility among strains, usually running equal to or faster than chromosome 1 but in rare cases running slower than chromosome 1. In strain 1012A, the decreased mobility of one homolog is associated with the very large majority of the rDNA genes being on that homolog; the second homolog, with only a few copies, migrates with chromosome 2. Linkage analysis by using spheroplast fusion confirms the gene assignments made by hybridization to blots of the electrophoretic karyotype. A newly cloned gene, LYS2, hybridizes to chromosome 1.

Candida dubliniensis and Candida albicans, the most common human fungal pathogen, have most of the same genes and high sequence similarity, but C. dubliniensis is less virulent. C. albicans causes both mucosal and hematogenously disseminated disease, C. dubliniensis mostly mucosal infections. Pulse-field electrophoresis, genomic restriction enzyme digests, Southern blotting, and the emerging sequence from the Wellcome Trust Sanger Institute were used to determine the karyotype of C. dubliniensis type strain CD36. Three chromosomes have two intact homologues. A translocation in the rDNA repeat on chromosome R exchanges telomere-proximal regions of R and chromosome 5. Translocations involving the remaining chromosomes occur at the Major Repeat Sequence. CD36 lacks an MRS on chromosome R but has one on 3. Of six other C. dubliniensis strains, no two had the same electrophoretic karyotype. Despite extensive chromosome rearrangements, karyotypic differences between C. dubliniensis and C. albicans are unlikely to affect gene expression. Karyotypic instability may account for the diminished pathogenicity of C. dubliniensis.

Background: The 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined. Results: Supercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome. Conclusion: Assembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution., National Institute of Allergy and Infectious Diseases (U.S.) (contract N01 AI05406), National Institute of Allergy and Infectious Diseases (U.S.) (grant R01 AI 16567), National Research Council (U.S.). Genome Health Initiative, Canadian Institutes for Health Research (grant HOP 67260), Canadian Institutes for Health Research (grant MOP 42516), Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research)

The release of the diploid genomic sequence of Candida albicans and its recent community-based annotation have permitted a number of studies which have significantly advanced our understanding of the biology of this important human pathogen. These advances range from analysis of genomic changes to differential gene expression under a variety of conditions. A few general conclusions can be drawn from the data presently in hand; one can expect more and more new insights as the number and kind of experiments grows.

Single-nucleotide polymorphisms (SNPs) are essential tools for studying a variety of organismal properties and processes, such as recombination, chromosomal dynamics, and genome rearrangement. This paper describes the development of a genome-wide SNP map for Candida albicans to study mitotic recombination and chromosome loss. C. albicans is a diploid yeast which propagates primarily by clonal mitotic division. It is the leading fungal pathogen that causes infections in humans, ranging from mild superficial lesions in healthy individuals to severe, life-threatening diseases in patients with suppressed immune systems. The SNP map contains 150 marker sequences comprising 561 SNPs and 9 insertions-deletions. Of the 561 SNPs, 437 were transition events while 126 were transversion events, yielding a transition-to-transversion ratio of 3:1, as expected for a neutral accumulation of mutations. The average SNP frequency for our data set was 1 SNP per 83 bp. The map has one marker placed every 111 kb, on average, across the 16-Mb genome. For marker sequences located partially or completely within coding regions, most contained one or more nonsynonymous substitutions. Using the SNP markers, we identified a loss of heterozygosity over large chromosomal fragments in strains of C. albicans that are frequently used for gene manipulation experiments. The SNP map will be useful for understanding the role of heterozygosity and genome rearrangement in the response of C. albicans to host environments.

One hundred and twenty Candida albicans clinical isolates from the late 1980s and early 1990s were examined for homozygosity at the MTL locus. Of these, 108 were heterozygous (MTLa/MTLalpha), whereas seven were MTLa and five were MTLalpha. Five of the homozygous isolates were able to switch to the opaque cell morphology, while opaque cells were not detectable among the remaining seven. Nevertheless, all but one of the isolates homozygous at the MTL locus were shown to mate and to yield cells containing markers from both parents; the non-mater was found to have a frameshift in the MTLalpha1 gene. In contrast to Saccharomyces cerevisiae, C. albicans homozygotes with no active MTL allele failed to mate rather than mating as a cells. There was no correlation between homozygosity and fluconazole resistance, mating and fluconazole resistance or switching and fluconazole resistance, in part because most of the strains were isolated before the widespread use of this antifungal agent, and only three were in fact drug resistant. Ten of the 12 homozygotes had rearranged karyotypes involving one or more homologue of chromosomes 4, 5, 6 and 7. We suggest that karyotypic rearrangement, drug resistance and homozygosity come about as the result of induction of hyper-recombination during the infection process; hence, they tend to occur together, but each is the independent result of the same event. Furthermore, as clinical strains can mate and form tetraploids, mating and marker exchange are likely to be a significant part of the life cycle of C. albicans in vivo.

We present the diploid genome sequence of the fungal pathogen Candida albicans . Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N -acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway. Fil: Cloutier, Monikca. GREB Laval University; Canadá Fil: Castilla Lozano, Maria del Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Bolduc, Nathalie. GREB Laval University; Canadá Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Martineau, Philippe. GREB Laval University; Canadá Fil: Bouillon, Marlène. GREB Laval University; Canadá Fil: Magee, Beatrice B.. University of Minnesota; Estados Unidos Fil: Di Bernardo, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina Fil: Giasson, Luc. GREB Laval University; Canadá Fil: Cantore, Maria Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Invetigaciones Bioquímicas y Fisiologicas; Argentina

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

Candida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.

We assessed the exchange of Candida albicans among crew members during 10 Space Shuttle missions. Throat, nasal, urine and faecal specimens were collected from 61 crew members twice before and once after space flights ranging from 7 to 10 days in duration; crews consisted of groups of five, six or seven men and women. Candida albicans was isolated at least once from 20 of the 61 subjects (33%). Candida strains were identified by restriction-fragment length polymorphism (RFLP) after digestion by the endonucleases EcoRI and HinfI; further discrimination was gained by Southern blot hybridization with the C. albicans repeat fragment 27A. Eighteen of the 20 Candida-positive crew members carried different strains of C. albicans in the specimens collected. Possible transfer of C. albicans between members of the same crew was demonstrated only once in the 10 missions studied. We conclude that the transfer of C. albicans among crew members during Space Shuttle flights is less frequent than had been predicted from earlier reports.

The opportunistic fungal pathogen, Candida albicans, is diploid as usually isolated and has no apparent sexual cycle. Genetic analysis has therefore been very difficult. Molecular genetics has yielded important information in the past few years, but it too is hampered by the lack of a good genetic map. Using the well-characterized strain 1006 and strain WO-1, which undergoes the white-opaque phenotypic transition, we have developed a genomic restriction map of C. albicans with the enzyme SfiI. There are approximately 34 SfiI restriction sites in the C. albicans genome. Restriction fragments were separated by pulsed-field electrophoresis and were assigned to chromosomes by hybridization of complete and partial digests with known chromosome-specific probes as well as by digestion of isolated chromosomes. Telomeric fragments were identified by hybridization with a telomere-specific probe (C. Sadhu, M.J. McEachern, E.P. Rustchenko-Bulgac, J. Schmid, D.R. Soll, and J.B. Hicks, J. Bacteriol. 173:842-850, 1991). WO-1 differs from 1006 in that it has undergone three reciprocal chromosomal translocations. Analysis of the translocation products indicates that each translocation has occurred at or near an SfiI site; thus, the SfiI fragments from the two strains are similar or identical. The tendency for translocation to occur at or near SfiI sites may be related to the repeated sequence RPS 1, which contains four such sites and could provide homology for ectopic pairing and crossing over. The genome size of both strains is about 16 to 17 megabases, in good agreement with previous determinations.

The secreted aspartyl proteinase activity from Candida albicans is thought to be a potential virulence factor. Four laboratories have cloned a gene from C. albicans encoding this enzyme. When two of these genes sharing 77% homology at the DNA level are hybridized under conditions of high stringency to contour-clamped homogeneous electric field chromosome separations of four different strains, they label different chromosomes: chromosome 6 for SAP1 and chromosome R for SAP2. The existence of different genes for the two sequences was confirmed by polymerase chain reaction. Genomic Southern blots probed with the genes and washed at low stringency revealed several cross-hybridizing bands. Contour-clamped homogeneous electric field chromosome separations probed at low stringency indicated that there was a cross-hybridizing sequence on chromosome 3 in addition to those on chromosomes R and 6. The genes for the secreted aspartyl proteinase activity in C. albicans thus constitute a gene family which we have called the SAP family.

Pulsed-field gel electrophoresis was used to analyze several aspects of the genetics of Candida albicans. A restriction map of the genome of strain 1006 was prepared using the eight-base-pair specific enzyme SfiI. The previously determined genetic map was superimposed on this physical map. Preparation of a similar map for the white-opaque strain WO-1 demonstrated the presence of three reciprocal chromosomal translocations in this organism. Mapping the cloned genes for the extracellular aspartyl proteinase to the electrophoretic karyotype under different stringencies demonstrated that there are at least four genes homologous to both the cloned genes.

Epidemiologic studies in women with recurrent Candida vaginitis have been hampered in the past by the lack of a reproducible typing system. Several molecular probes have now been developed that have the ability to differentiate strains of Candida albicans and give reproducible results. In this investigation, 24 women with Candida vaginitis were studied in a longitudinal fashion for 30 days following short-course antifungal therapy. Seven women with either recurrent vaginitis or with multiple culture-positive sites with C. albicans were included in an epidemiological study. A total of 18 isolates of C. albicans (12 vaginal and six rectal) were typed utilizing restriction fragment length polymorphisms of rDNA. This technique was able to differentiate five different strains of C. albicans. Our epidemiologic study revealed that vaginal and rectal strains recovered from the same women were usually different. None of our patients had a similar vaginal and rectal strain prior to treatment, and only one patient had the same strain isolated from both the rectum and the vagina at the time of recurrence. On the other hand, we found that the same strain of C. albicans was initially and later recovered from the vagina in four of five women who failed treatment or developed recurrent vaginitis. These results suggest that recurrent episodes of C. albicans vaginitis, following short-course antifungal therapy, are often due to relapse of the original infecting strain and not due to autoinoculation from the rectum.(ABSTRACT TRUNCATED AT 250 WORDS)

Candida albicans and Candida stellatoidea are two closely related imperfect yeasts. Some isolates characterized as C. stellatoidea are in fact C. albicans, while others differ with respect to virulence and to karyotype, containing extra small chromosomes. Experiments in this study allowed us to infer that a typical C. stellatoidea isolate, Y2360, has 12 chromosomes rather than the 7 previously shown for C. albicans. The majority of cloned sequences tested hybridized to analogous chromosomes in C. albicans and in C. stellatoidea, although there were exceptions, and a repeated element isolated as specific for C. albicans hybridized to most of the chromosomes of C. stellatoidea. Several genes tested hybridized to one of the smaller, C. stellatoidea-specific chromosomes as well as to a larger one. The arrangement of restriction enzyme sites around the gene was the same in both the large and small chromosomes. For ADE2 and LYS2, the arrangements were identical to those of a typical C. albicans strain, FC18, suggesting a high degree of sequence conservation between the two species. Spheroplast fusion and segregation experiments showed that the ADE2 genes on both the large and small chromosomes of C. stellatoidea are active, implying that the organism is functionally at least triploid for this gene and probably for any others duplicated on the smaller chromosomes.

This paper reports that the opaque and white phenotypes of Candida albicans constitute a true high-frequency reversible transition system. The rDNA restriction fragment and orthogonal field alternating gel electrophoresis profiles of opaque and white phenotypes are indistinguishable, and a genetic marker introduced into a white strain is present in all opaque derivatives of this strain. Opaque and white derivatives appear markedly different on a bismuth indicator medium and differ in a number of other respects. We have used bismuth medium to examine the spontaneous and temperature-induced frequencies of transition from opaque to white. The temperature-induced transition from opaque to white does not occur when opaque cells are held in water.

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

Chromosomes from Cryptococcus neoformans, an encapsulated yeast pathogen, were separated by contour-clamped homogeneous field gel electrophoresis. Seven strains representing all four serotypes were studied. It was found that each strain had a unique, reproducible pattern of chromosome bands which could potentially be used for strain polymorphism studies. There were between 10 and 12 chromosomes in the strains studied, with an approximate genomic size of 15,000 to 17,000 kilobases. Chromosome separation also could be used to assign locations for cloned genes, and the ribosomal DNA genes were found on one of the larger C. neoformans chromosomes. The technique of electrophoretic karyotyping should be helpful for genetic and molecular investigations into the biology of C. neoformans.

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

The electrophoretic karyotypes of five Candida albicans isolates and of five other Candida species have been determined, using orthogonal field alternating gel electrophoresis (OFAGE). None of the C. albicans isolates had the same electrophoretic karyotype. By comparing all five strains, we arrived at a chromosome number of nine to ten, but since the organism is diploid, we cannot distinguish genetically different chromosomes from homologues which resolve. We determined minimal chromosome numbers of 9 for Candida stellatoidea, 10 for C. glabrata and 6 for C. guilliermondii.

Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.

Genetic similarities and differences between type I and type II Candida stellatoidea were studied. The electrophoretic karyotype, mitochondrial DNA (mtDNA) restriction patterns, and midrepeat sequence of nuclear DNA in type I C. stellatoidea were clearly distinguishable from those of a reference culture of Candida albicans. The karyotype and the major bands of the midrepeat sequence of type II C. stellatoidea were indistinguishable from those of the reference C. albicans. The mtDNA restriction patterns of four type I isolates were homogeneous regardless of the endonucleases and probes used. The mtDNA restriction patterns of type II C. stellatoidea varied from strain to strain. Some of them were identical to that of C. albicans, while others were the same as that of type I C. stellatoidea. Immunofluorescence with C. albicans serotype A-specific monoclonal antibody indicated that the four isolates of type I C. stellatoidea were serotype B (non-A), whereas all three type II isolates studied were serotype A. Taken together, these results support the hypothesis that the isolates of C. stellatoidea type II studied are sucrose-negative mutants of serotype A C. albicans. Since C. stellatoidea type I differs from C. albicans in several major genetic characteristics, it cannot be viewed as a simple mutant derived from C. albicans. Hybrids produced by protoplast fusion of type I and type II cells were capable of assimilating sucrose, indicating that the sucrose-negative phenotypes of the parents are due to different mutations.

Background Invasion of host tissue by the human fungal pathogen Candida albicans is an important step during the development of candidosis. However, not all C. albicans strains possess the same invasive and virulence properties. For example, the two clinical isolates SC5314 and ATCC10231 differ in their ability to invade host tissue and cause experimental infections. Strain SC5314 is invasive whereas strain ATCC10231 is non-invasive and strongly attenuated in virulence compared to SC5314. In this study we compare the in vitro phenotypic, transcriptional and genomic profiles of these two widely used laboratory strains in order to determine the principal biological and genetic properties responsible for their differential virulence. Results In all media tested, the two strains showed the same metabolic flexibility, stress resistance, adhesion properties and hydrolytic enzyme secretion in vitro. However, differences were observed in response to cell-surface disturbing agents and alkaline pH. Furthermore, reduced hyphal formation in strain ATCC10231 under certain conditions correlated with reduced invasive properties in an in vitro invasion assay and a reduced ability to invade epithelial tissue. Despite these diverse phenotypic properties, no substantial genomic differences were detected by comparative genome hybridisation within the open reading frames. However, in vitro transcriptional profiling displayed major differences in the gene expression of these two strains, even under normal in vitro growth conditions. Conclusion Our data suggest that the reason for differential virulence of C. albicans strains is not due to the absence of specific genes, but rather due to differences in the expression, function or activity of common genes.