Beatrice B. Magee

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

BackgroundThe 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.ResultsSupercontigs 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.ConclusionAssembly 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.

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans.

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 MTLα strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mfα, prevented mating in MTLα 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 MTLα 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.

Homozygosity at the MTL locus in clinical strains of Candida albicans: karyotypic rearrangements and tetraploid formation †

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.

Chromosome 1 trisomy compromises the virulence of Candida albicans

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 Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

ABSTRACT 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.

Genome-Wide Single-Nucleotide Polymorphism Map for Candida albicans

ABSTRACT 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.

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans

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.

A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae

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 ste6Δ 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‐Matα2p 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.

Cloning, sequencing and chromosomal assignment of a gene from Saccharomyces cerevisiae which is negatively regulated by glucose and positively by lipids

We report the molecular cloning, nucleotide (nt) sequence and chromosomal assignment of the Saccharomyces cerevisiae gene GLP1. This gene encoded a 15-kDa protein that was synthesized at a low level during growth on glucose and was induced ninefold upon glucose deprivation. When glucose withdrawal was accompanied by the addition of fatty acids the induction was enhanced an additional two- to threefold. The GLP1 gene product was identified as a soluble protein and purified using a combination of gel permeation and ion exchange chromatography. Using oligodeoxyribonucleotides as hybridization probes we have isolated the GLP1 gene and sequenced the single, long open reading frame which is 351 nt in length and is not interrupted by introns. The GLP1 gene directed the transcription of a 700-nt mRNA in response to glucose deprivation. The accumulation of the mRNA was further enhanced twofold by the addition of oleate. We have localized the GLP1 gene to S. cerevisiae chromosome VI.

Phenotypic screening, transcriptional profiling, and comparative genomic analysis of an invasive and non-invasive strain of Candida albicans

BackgroundInvasion 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.ResultsIn 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.ConclusionOur 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.