Homothallic and heterothallic mating in the opportunistic pathogen Candida albicans.

Candida albicans is the most common fungal pathogen in humans, causing both debilitating mucosal infections and potentially life-threatening systemic infections. Until recently, C. albicans was thought to be strictly asexual, existing only as an obligate diploid. A cryptic mating cycle has since been uncovered in which diploid a and alpha cells undergo efficient cell and nuclear fusion, resulting in tetraploid a/alpha mating products. Whereas mating between a and alpha cells has been established (heterothallism), we report here two pathways for same-sex mating (homothallism) in C. albicans. First, unisexual populations of a cells were found to undergo autocrine pheromone signalling and same-sex mating in the absence of the Bar1 protease. In both C. albicans and Saccharomyces cerevisiae, Bar1 is produced by a cells and inactivates mating pheromone alpha, typically secreted by alpha cells. C. albicans Deltabar1 a cells were shown to secrete both a and alpha mating pheromones; alpha-pheromone activated self-mating in these cells in a process dependent on Ste2, the receptor for alpha-pheromone. In addition, pheromone production by alpha cells was found to promote same-sex mating between wild-type a cells. These results establish that homothallic mating can occur in C. albicans, revealing the potential for genetic exchange even within unisexual populations of the organism. Furthermore, Bar1 protease has an unexpected but pivotal role in determining whether sexual reproduction can potentially be homothallic or is exclusively heterothallic. These findings also have implications for the mode of sexual reproduction in related species that propagate unisexually, and indicate a role for specialized sexual cycles in the survival and adaptation of pathogenic fungi.

The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains.

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and alpha strains. The product of mating is a tetraploid a/alpha cell that must undergo a reductional division to return to the diploid state. Despite the presence of several "meiosis-specific" genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.

Identification of a cell death pathway in Candida albicans during the response to pheromone.

Mating in hemiascomycete yeasts involves the secretion of pheromones that induce sexual differentiation in cells of the opposite mating type. Studies in Saccharomyces cerevisiae have revealed that a subpopulation of cells experiences cell death during exposure to pheromone. In this work, we tested whether the phenomenon of pheromone-induced death (PID) also occurs in the opportunistic pathogen Candida albicans. Mating in C. albicans is uniquely regulated by white-opaque phenotypic switching; both cell types respond to pheromone, but only opaque cells undergo the morphological transition and cell conjugation. We show that approximately 20% of opaque cells, but not white cells, of laboratory strain SC5314 experience pheromone-induced death. Furthermore, analysis of mutant strains revealed that PID was significantly reduced in strains lacking Fig1 or Fus1 transmembrane proteins that are induced during the mating process and, we now show, are necessary for efficient mating in C. albicans. The level of PID was also Ca(2+) dependent, as chelation of Ca(2+) ions increased cell death to almost 50% of the population. However, in contrast to S. cerevisiae PID, pheromone-induced killing of C. albicans cells was largely independent of signaling via the Ca(2+)-dependent protein phosphatase calcineurin, even when combined with the loss of Cmk1 and Cmk2 proteins. Finally, we demonstrate that levels of PID vary widely between clinical isolates of C. albicans, with some strains experiencing close to 70% cell death. We discuss these findings in light of the role of prodeath and prosurvival pathways operating in yeast cells undergoing the morphological response to pheromone.

Partner choice during meiosis is regulated by Hop1-promoted dimerization of Mek1.

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.

Barrier activity in Candida albicans mediates pheromone degradation and promotes mating.

Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Deltabar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in alpha cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Deltabar1 a and alpha cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.