Evolution in Candida albicans populations during a single passage through a mouse host.

The mechanisms and rates by which genotypic and phenotypic variation is generated in opportunistic, eukaryotic pathogens during growth in hosts are not well understood. We evaluated genomewide genetic and phenotypic evolution in Candida albicans, an opportunistic fungal pathogen of humans, during passage through a mouse host (in vivo) and during propagation in liquid culture (in vitro). We found slower population growth and higher rates of chromosome-level genetic variation in populations passaged in vivo relative to those grown in vitro. Interestingly, the distribution of long-range loss of heterozygosity (LOH) and chromosome rearrangement events across the genome differed for the two growth environments, while rates of short-range LOH were comparable for in vivo and in vitro populations. Further, for the in vivo populations, there was a positive correlation of cells demonstrating genetic alterations and variation in colony growth and morphology. For in vitro populations, no variation in growth phenotypes was detected. Together, our results demonstrate that passage through a living host leads to slower growth and higher rates of genomic and phenotypic variation compared to in vitro populations. Results suggest that the dynamics of population growth and genomewide rearrangement contribute to the maintenance of a commensal and opportunistic life history of C. albicans.

Induction of mating in Candida albicans by construction of MTLa and MTLalpha strains.

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 alpha. When strains containing only MTLa or MTLalpha were constructed by the loss of one homolog of chromosome 5, the site of the MTL loci, MTLa and MTLalpha 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.

Extensive chromosome rearrangements distinguish the karyotype of the hypovirulent species Candida dubliniensis from the virulent Candida albicans.

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.

Candida pathogenesis: unravelling the threads of infection.

Recent studies are beginning to delineate those pathways by which the important pathogen Candida albicans switches from one growth form to another; at the same time, insights are being gained into the importance of growth form in pathogenesis.

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

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.

Sequence finishing and gene mapping for Candida albicans chromosome 7 and syntenic analysis against the Saccharomyces cerevisiae genome.

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.

Through a glass opaquely: the biological significance of mating in Candida 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.

Variations in chromosome size and organization in Candida albicans and Candida stellatoidea.

Candida albicans and the closely related species Candida stellatoidea are medically important diploid asexual yeasts. Clinical isolates frequently show variant electrophoretic karyotypes, apparently due largely to chromosomal translocations. These translocations seem to occur at hot spots characterized by the repeated DNA sequence RSP1. A programmed karyotypic rearrangement occurs in C. stellatoidea. Karyotypic rearrangement may serve as a source of genetic variation in these asexual yeasts.

Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae.

Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells. The partially purified enzyme was active against glycogen, starch, maltotriose and maltose. It exhibited maximum catalytic activity against glycogen at pH 5.5. The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose. SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques. SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.

Identification and characterization of mutations affecting sporulation in Saccharomyces cerevisiae.

Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1. Two were found, both of which were deficient in sporulation at 34 degrees. One, SL484, sporulated to 50% normal levels at 30 degrees but less than 5% at 34 degrees or 22 degrees. The other, SL641, failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis, and both complemented spo1, spo3, and spo7. Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII. A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18. These two genes are (with SPO7, SPO8, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.

A genetic analysis of Candida albicans: isolation of a wide variety of auxotrophs and demonstration of linkage and complementation.

Naturally occurring strains of Candida albicans appear to be diploid and heterozygous for a limited number of nutritional markers. Additional heterozygosity can be induced by treatment with mutagens; nitrous acid alone or in combination with UV is a potent mutagen in terms of both efficacy and efficiency in the production of a wide variety of mutations. Spheroplast fusion followed by regeneration on selective media revealed complementation among four histidine-requiring mutants analyzed. Some of the fusion products appeared to be stable prototrophs, whereas in others several kinds of segregants resulted, apparently due to chromosomal or nuclear elimination. The results are suggestive of both heterokaryosis as well as nuclear fusion. The procedures described can be successfully used for generating new mutants and studying allelism. Three sets of linkage relationships have been derived from evidence provided by concomitant appearance or cosegragation of several auxotrophic markers.

A physical map of chromosome 7 of Candida albicans.

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.

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.

Purification and properties of threonine deaminase from Saccharomyces cerevisiae.

Threonine deaminase (L-theonine hydro-lyase (deaminating), E.C. 4.2.1.16) has been purified to homogeneity from extracts of Saccharomyces cerevisiae. When purified 1200-fold, the enzyme is homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide electrophoresis. The reduced and alkylated protein has a molecular weight of approximately 50,000 daltons, one-fourth the value determined previously for the intact enzyme. The purified enzyme exhibits homotropic effects with the substrate; these effects are descresed in the presence of DL-allothreonine, a competitive inhibitor. Half-maximal velocity is achieved at 34 mM L-threonine in the absence of other effectors. L-isoleucine both stimulates at low (0.01-0.05 mM) concentrations and inhibits at high (0.1-1.0 mM) concentrations. Valine activates the enzyme in the absence of isoleucine ; in the presence of isoleucine it reverses inhibition.

Use of rDNA restriction fragment length polymorphisms to differentiate strains of Candida albicans in women with vulvovaginal candidiasis.

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)

Fungal pathogenicity and morphological switches.

The virulence of Candida albicans, a major human fungal pathogen, has been considered dependent on the ability to transition between different morphologies. A new study reports a screen of C. albicans mutants that demonstrates that pathogenesis can be dissociated from morphological switching and in vitro growth rate.