Completion of a parasexual cycle in Candida albicans by induced chromosome loss in tetraploid strains

Abstract

The human pathogenic fungus Candida albicans has traditionally been classified as a diploid, asexual organism. However, mating-competent forms of the organism were recently described that produced tetraploid mating products. In principle, the C.albicans life cycle could be completed via a sexual process, via a parasexual mechanism, or by both mechanisms. Here we describe conditions in which growth of a tetraploid strain of C.albicans on Saccharomyces cerevisiae 'pre-sporulation' medium induced efficient, random chromosome loss in the tetraploid. The products of chromosome loss were often strains that were diploid, or very close to diploid, in DNA content. If they inherited the appropriate MTL (mating-type like) loci, these diploid products were themselves mating competent. Thus, an efficient parasexual cycle can be performed in C.albicans, one that leads to the reassortment of genetic material in this organism. We show that this parasexual cycle-consisting of mating followed by chromosome loss-can be used in the laboratory for simple genetic manipulations in C.albicans.

Fig. 1. Putative sexual cycle of C.albicans. Mating between a and α opaque cells produces an a/α tetraploid cell. A reduction in ploidy back to the diploid state, which could in principle occur by random chromosome loss or by meiosis, is the subject of this paper.

Fig. 2. Test of stability of a tetraploid strain grown on different media. (A and B) The tetraploid strain used in this study (RBY18) is hetero zygous at the GAL1, ADE2 and MTL loci on chromosomes 1, 3 and 5, respectively. (C) The tetraploid strain was grown for 8 days on different media at 22, 30 or 37°C, respectively. Cells were pooled from the different media and plated on FOA/DOG medium and YEPD. The number of cells that could grow on the FOA/DOG plates relative to YEPD was calculated (number of colonies per 10⁵ cells). (D) The tetraploid strain was grown on YEPD or pre-spo medium for 8 days at 37°C. Following incubation, cells were recovered and tested for growth on several test media. The percentage of cells that could grow on the test plates (or gave red colonies indicating Ade– colonies) relative to YEPD was calculated.

Fig. 3. FACS analysis of tetraploid strains that had been exposed to pre-spo medium. Single colonies were selected and grown up in liquid YEPD medium for FACS analysis, as described in Materials and methods. The x-axis of each graph (Sytox) represents a linear scale of fluorescence, and the y-axis (Counts) represents a linear scale of cell number. (A) Comparison of a control diploid strain (CAF2-1; left trace) with the starting tetraploid strain (RBY18; right trace). Control diploid and tetraploid traces are also included for reference in (B–F) and (H–K). (B–F) Five independent colonies (PS1–PS5) derived from the tetraploid strain RBY18 after incubation on pre-spo medium; all five colonies were di-allelic at MTL. (G) Comparison of the FACS profiles of four different laboratory diploid strains, CAF2-1, CHY257, CHY439 and CHY477 (Miller and Johnson, 2002). (H and I) Two colonies derived from the tetraploid strain after growth on pre-spo medium: these were chosen for FACS analysis because they were tri-allelic at the MTL locus. (J and K) Two colonies that were still tetra-allelic at MTL after incubation on pre-spo medium.

Fig. 4. Pre-spo medium induces allele loss in a tetraploid strain of C.albicans. (A) A detailed analysis of growth of the tetraploid strain on pre-spo medium was carried out. The tetraploid strain RBY18 was grown for 8 days at 37°C on pre-spo medium and pooled colonies were subsequently tested for growth on DOG or YEPD plates. The percentage of cells that were DOG^R, Ade– or di-allelic at the MTL locus is indicated (1° screen). The DOG^R and Ade– colonies were further tested to determine the percentage of these cells that had lost other chromosomal markers (2° screen). (B) The tetraploid strain was grown on YEPD for 8 days at 37°C, and subsequently analyzed as in (A). The results of (A) and (B) show that allele loss is concerted.

Fig. 5. Analysis of the MTL locus in cells grown on pre-spo medium. The configuration of the MTL was checked by PCR after tetraploid cells had been exposed to pre-spo medium at 37°C for 8 days. (A) Schematic representation of the PCR products used to identify the different MTL alleles. Black bands represent the position of the relevant PCR products, while gray bands represent non-specific products amplified in the PCR reaction. Each reaction contains three sets of primers and, in this ideal diagram, all four MTL alleles (a, α, Δa, Δα) are present in the strain. (B) PCR products from six different progeny colonies are shown, together with a tetraploid control. All six possible MTL configurations were observed, indicating the random nature of the chromosome loss.

Fig. 6. Time course of chromosome loss and viability. (A) The tetraploid strain, RBY18, was grown on pre-spo (solid lines) or YEPD (dashed lines) medium at 37°C. Cells were collected at regular intervals and plated onto FOA, DOG or YEPD plates. The frequency of allele loss is proportional to the percentage of cells that grew on FOA or DOG plates relative to YEPD control plates, and by the percentage of Ade– (red) colonies on YEPD. (B) The percentage of viable cells was calculated by collecting cells, measuring the total OD₆₀₀ and then plating a fixed number of cells onto YEPD plates.

Fig. 7. Growth on sorbose medium induces chromosome loss from tetra ploids. The tetraploid strain RBY18 was grown on sorbose medium at 37°C for 7 days. (A) Chromosome loss was monitored by calculating the percentage of cells that were DOG^R, Ade– or di-allelic at MTL (1° screen). DOG^R colonies were also tested to see what percentage were Ade– or di-allelic at MTL, and Ade– colonies similarly tested to find the percentage that were DOG^R or di-allelic at MTL (2° screen). (B) Four colonies picked from the sorbose medium and determined to be di-allelic at MTL were grown up in YEPD and analyzed by FACS. In each graph, a control diploid strain, the parent tetraploid strain and the test colony are included (see Figure 3 for details).

Fig. 8. Chromosome loss in a tetraploid can be used to link a phenotype with a genotype. A tetraploid strain that is heterozygous for the NRG1 gene (nrg1/nrg1/NRG1/NRG1) was constructed by mating two Δnrg1/NRG1 strains, and then exposed to pre-spo medium to induce chromosome loss. (A) Both smooth and wrinkled colonies were obtained following chromosome loss on pre-spo. A control nrg1/nrg1 diploid strain is shown with its characteristic wrinkled appearance. Colony PCR was carried out on smooth colonies (B) or wrinkly colonies (C) generated from the tetraploid exposed to pre-spo medium in order to detect the presence or absence of the NRG1 ORF. Only smooth colonies showed the presence of the NRG1 gene. This analysis shows that the wrinkled appearance is caused by the NRG1 deletion (or a mutation closely linked to it). (Upper PCR bands are background bands from the PCR reaction.)

Fig. 9. Chromosome loss induced by pre-spo medium can be used to demonstrate genetic linkage. (A) Two mating experiments were carried out in which a gal1 hop1 strain was crossed with a GAL1 HOP1 strain (cross 1), or a gal1 HOP1 strain was crossed with a GAL1 hop1 strain (cross 2). The resultant tetraploid strains were incubated on pre-spo medium at 37°C to induce chromosome loss. If the GAL1 and HOP1 genes are linked, all the DOG^R (i.e. gal1–) progeny from cross 1 will lack the HOP1 gene, while all the DOG^R progeny from cross 2 will contain the wild-type HOP1 gene. (B) Schematic of the PCR used to test whether a colony contains the wild-type HOP1 gene or the hop1 deletion. (C) Colony PCR was performed on 14 DOG^R progeny from cross 1 (lanes 1–14) and 14 DOG^R progeny from cross 2 (lanes 15–28). All PCR reactions contained primer sets to detect both the deleted and wild-type HOP1 alleles. The results show that HOP1 is genetically linked to GAL1.