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Induction of Mating in Candida albicans by Construction of MTLa and MTLα Strains

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Science  14 Jul 2000:
Vol. 289, Issue 5477, pp. 310-313
DOI: 10.1126/science.289.5477.310

Abstract

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 theSaccharomyces cerevisiae mating-type genes (MAT) a and α. When strains containing only MTL a or MTLα were constructed by the loss of one homolog of chromosome 5, the site of theMTL loci, MTL a and MTLα 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 MTLalleles and molecular markers from both parents and were tetraploid in DNA content and mononucleate.

C. albicans has become the third or fourth most common nosocomial isolate in many hospitals, and systemic infection has a mortality rate of 30 to 70% (1). This yeast is almost always diploid as isolated (2) and until now was thought to lack a sexual phase (3).

Several hypotheses have been put forward for the way in which C. albicans generates genetic variability, including phenotypic transition, or switching, extensively studied in the Soll laboratory (4), and chromosomal rearrangements (5,6). Both are associated with changes in the phenotype, but would be expected to yield less variability than sexual recombination.

Evidence for asexuality in C. albicans consists of the failure of many laboratories to find either mating or sporulation, the frequent isolation of strains with extensive chromosomal translocations that would lead to aneuploid progeny after meiosis (7,8), and evidence for linkage disequilibrium, typical of clonal inheritance (9).

However, with the sequencing of the C. albicans genome, evidence has emerged suggesting that sexual recombination has occurred relatively recently in this fungus. This evidence includes the discovery of orthologs of most, if not all, of the genes involved in mating and sporulation in Saccharomyces cerevisiae, including genes for pheromone receptors and heterotrimeric GTP-binding proteins, and specifically the demonstration, with information from the Stanford Genome Center Candida albicans Sequencing Project (10), that at least one strain of C. albicansis heterozygous for orthologs of the two S. cerevisiaemating types MAT a and MATα. These loci are called mating-type–like (MTL)a and α in C. albicans (11). There is also evidence for a lack of linkage of some molecular chromosomal markers (12).

Taking advantage of the location of the MTL locus on a chromosome, 5, that can be reduced to monosomy by changing the simple growth conditions (13), we constructed several sets ofC. albicans strains that lack either the a or the α MTL allele; these strains mated in a-α combinations to yield tetraploid recombinants.

C. albicans is normally unable to grow on sorbose (Sou). The Sou+ variants, which appear on the background of nongrowing cells plated on sorbose as a sole source of carbon, are monosomic for chromosome 5; furthermore, either homolog can be lost under these conditions (13). Using this technique, we isolated Sou+ derivatives of strains CAI-4, RM1000, BWP17, B23, and B26 lacking either MTLa orMTLα as shown by Southern blots (14) (Fig. 1A, lanes 3, 5, 7, 9, 10, 12, and 13). Table 1 summarizes the properties of the strains used in the experiments described below.

Figure 1

Molecular genomic markers ofC. albicans strains in mating experiments. (A) Southern transfers of the pulsed-field gel (31) were probed with PCR products from MTL a and α. All of the parental strains were heterozygous at the MTL locus and had both a and α sequences, whereas the Sou+variants had one allele or the other. Lane 1, 1006 (7); lane 2, 3199; lane 3, 3251; lane 4, 3205; lane 5, 3252; lane 6, 3206; lane 7, 3253; lane 8, 3312; lane 9, 3270; lane 10, 3271 (MTL a derivative of 3312); lane 11, 3313; lane 12, 3272; lane 13, 3273; lane 14, X32; lane 15, X40. The numbers refer to the strains listed in Table 1. (B) DNA of parents and progeny was digested with Eco RI, blotted, and probed with PCR products from MTL a and α and from the λimm insert in CAI-4 (3199). Parental strains derived from CAI-4 carry this insert and are Ura, whereas those from MG30 are Ura+ and do not have the insert. The recombinants are Ura+, and all but two also contain the insert. Lane 1, 3251; lane 2, 3252; lane 3, 3253; lane 4, X1; lane 5, X2; lane 6, X12; lane 7, X13; lane 8, X14; lane 9, X21; lane 10, X23; lane 11, X28; lane 12, X32; lane 13, X40; lane 14, 1 kb size markers; lane 15, 3272; lane 16, 3273; lane 17, 3313; lane 18, 3270; lane 19, 3312; lane 20, MG30. (C) The DNA of parents and progeny was digested with Hinf I, separated by gel electrophoresis, blotted, and probed with plasmid prDNA, containing a ribosomal DNA repeat.

Table 1

C. albicans strains used in mating experiments. Strains are listed by their numbers in the Scherer-Magee strain collection at the University of Minnesota. Original designations are listed for several strains: CAI-4 (17), RM1000 (28), BWP17 (29), and MG30 (30).

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If strains containing only one allele of the MTL locus are mating-competent, selectable prototrophic conjugants should arise from complementation of the various auxotrophic markers in the two sets of strains. The hemizygotic strains (or homozygotic; under nonselective conditions the strains often duplicate the remaining homolog of chromosome 5 and become homozygous for the entire chromosome) listed in Table 1 were grown on YEPD (yeast extract–peptone–dextrose) (15) plates until they were in stationary phase. They were cross-streaked on YEPD, YPB (YEPD plus phloxine B), and PD (potato dextrose agar) plates, incubated for 36 hours, and replica-plated onto minimal medium. In each case, several prototrophic colonies grew from one of the cross-streaked areas where potential complementation products would be formed by ana × α mating. Prototrophs formed by mating of theMTL a × MTLα strains should contain both MTL alleles. The cells were restreaked on minimal medium. Polymerase chain reaction (PCR) revealed that the prototrophs possessed both MTL alleles (16) and that, although they were able to grow on medium lacking uridine, they contained the λimm434 fragment which replaces theURA3 locus in CAI4 (17, 18). Thus, the prototrophic cells contained genomic material from both parents (Fig. 1A, lanes 14 and 15).

More efficient mating was found to occur when the parents were allowed to remain in contact on nonselective medium for 3 to 12 days before replication onto minimal (selective) medium (19). Under this regimen, profuse growth occurred on the selective medium, in crosses between MTL a- MTLα strains but not between those of like mating type. More prototrophs were formed when mating plates were incubated at room temperature than at 30° or 37°C (Fig. 2A). Mating at any temperature is a slow process, with the yield of recombinants rising from a few after 4 days on nonselective medium to many at 8 days (Fig. 2B).

Figure 2

Temperature and time dependence of prototroph formation (19). (A) The mating plates were incubated at the indicated temperatures and then replicated to minimal plates. Strains: 80α, 3280; 81α, 3281; 70a, 3270; 51α, 3251; 52a, 3252; and 53a, 3253. (B) The mating plates were incubated at room temperature for 4 and 8 days after replication. Strains: 79a, 3279; 80α, 3280; 81α, 3281; 01α, WO-1Ura; 51α, 3251; and 53a, 3253. WO-1Ura is a derivative of the strain WO-1 isolated by Slutsky et al. (23).

To verify that the recombinants were genetically prototrophic and not the result of cross-feeding or necrophagy of dead cells at the cross-streak intersection, we restreaked them onto minimal medium. The resulting large colonies were chosen to prepare DNA. The small colonies were revertants of the MG30 derivatives, and one revertant (X28) was included in our further analysis. The recombinants were tested for genomic markers by Southern blotting of genomic DNA from the parents and the recombinants. The DNA of the parents contained sequences that hybridized with probes from onlyMTL a or α, depending on the strain, but all the putative recombinants, with the exception of X28, contained bothMTL alleles (Fig. 1B). All the recombinants, with the exception of X1, X14, and X28, contained the λimm434region (Fig. 1B); because all grew on medium lacking uridine, they must have the URA3 gene.

A third genomic difference between the parents is a restriction fragment length polymorphism in the nontranscribed spacer of the ribosomal DNA, which is located on the largest of the C. albicans chromosomes, R (20). Digestion with the restriction enzyme Hinf I gives a 4.9-kb (kilobase pair) fragment from the CAI-4–derived parents and a 5.2-kb fragment from the MG30-derived parents (Fig. 1C). Each of the complementation products, with the exception of X14 (which lacks the 5.2 kb band) and X28, contains Hinf I products of both sizes. Because the MTL locus is found on chromosome 5 and the URA3imm434 insertion) locus on chromosome 3, a significant amount of genetic material (at least one homolog of three of the eight chromosomes) derived from each parent is found in most of the prototrophs.

If the transformants arose by classical (one-to-one) mating, they would be expected to have tetraploid DNA content. Propidium iodide was used to stain the nuclei of the recombinants, the parents, and a haploid S. cerevisiae strain for analysis in a fluorescence-activated cell sorter (FACS) (21). As expected, the parents contained about twice the DNA content of the S. cerevisiae haploid. The recombinants, however, contained approximately twice as much DNA as the parents and about fourfold the DNA content of the haploid S. cerevisiae strain (Fig. 3). The forward light-scattering analysis indicated that the tetraploid cells were about the same size as the parents and hence were not aggregates of diploid cells (18). That not all the mating products contained a full tetraploid DNA content is consistent with the observations that some recombinants did not contain all the markers from both parents. Loss of some markers may have occurred during the mating process or in subsequent mitotic divisions.

Figure 3

FACS analysis of the DNA content of parental strains and mating products. DNA content (x axis) is in arbitrary units. The mating products: (Top row) Parental strains 3253, 3273, and S. cerevisiae haploid strain 16. (Second row) Recombination products X32 and X2.

This study showed that C. albicans strains that were homozygous (a or α) at the MTL locus formed genetic recombinants with strains of the other mating type. It is possible that homozygosity for other genes on chromosome 5 plays a role in the capacity to mate, but we have no evidence for this. The strains used were clinical isolates resembling each other in common molecular characters but unlikely to be genetically related. They have been in laboratory strain collections for several years, so the ability to mate seems to be stable under these conditions.

The recombinants formed by the mating process contain most if not all of the genetic material of both parents. This form of genetic exchange differs from the artificial process of spheroplast fusion in that it does not require removal of the cell wall; it requires that the parental strains be homozygous and different at the MTLlocus, and it yields strains that are approximately tetraploid, rather than containing several parental genomic complements. Thus, it seems to be highly analogous to the mating process in S. cerevisiae.

However, we do not know the details of the process, such as whether it involves mating pheromones, although genes for pheromone receptors have been found by the Candida albicans Genome Sequencing Project (10).

The frequency of occurence of mating in nature is also unknown. Monosomy of chromosome 5, resulting from growth on sorbose, is unlikely to occur in nature, but other kinds of stress may induce nondisjunction, leading to homozygosity at the MTL locus. That in vitro mating is favored at temperatures lower than the body temperature of the host is in accord with observations that genetic recombination in vivo is rare.

It now seems clear that the first step of sexual exchange, mating with the formation of recombinants with genetic complements from both parents, can occur when strains homozygous for opposite mating types are mixed. Although we do not know how recombinant cells return to the diploid state characteristic of clinical isolates, i.e., whether meiosis and sporulation occur (22), the discovery thatC. albicans contains genes orthologous to those of the sexual machinery of S. cerevisiae indicates this organism has a complete sexual cycle (22). It seems likely that this process may be an important part of the life cycle of C. albicans in vivo.

  • * To whom correspondence should be addressed. E-mail: ptm{at}biosci.cbs.umn.edu

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