Date Published: May 2, 2013
Publisher: Public Library of Science
Author(s): Jianmin Fu, Ian R. Morris, Brian L. Wickes, Tamara L. Doering.
Cryptococcus neoformans is a heterothallic fungal pathogen of humans and animals. Although the fungus grows primarily as a yeast, hyphae are produced during the sexual phase and during a process called monokaryotic fruiting, which is also believed to involve sexual reproduction, but between cells of the same mating type. Here we report a novel monokaryotic fruiting mechanism that is dependent on the cell cycle and occurs in haploid cells in the absence of sexual reproduction. Cells grown at 37°C were found to rapidly produce hyphae (∼4 hrs) and at high frequency (∼40% of the population) after inoculation onto hyphae-inducing agar. Microscopic examination of the 37°C seed culture revealed a mixture of normal-sized and enlarged cells. Micromanipulation of single cells demonstrated that only enlarged cells were able to produce hyphae and genetic analysis confirmed that hyphae did not arise from α-α mating or endoduplication. Cell cycle analysis revealed that cells grown at 37°C had an increased population of cells in G2 arrest, with the proportion correlated with the frequency of monokaryotic fruiting. Cell sorting experiments demonstrated that enlarged cells were only found in the G2-arrested population and only this population contained cells able to produce hyphae. Treatment of cells at low temperature with the G2 cell cycle arrest agent, nocodazole, induced hyphal growth, confirming the role of the cell cycle in this process. Taken together, these results reveal a mating-independent mechanism for monokaryotic fruiting, which is dependent on the cell cycle for induction of hyphal competency.
Cryptococcus neoformans is a basidiomycetous fungal pathogen of humans and animals that typically causes opportunistic infections in patients with cellular immune defects . Infection initiates in the lungs and frequently disseminates to the brain where it manifests as a fatal meningoencephalitis if untreated. AIDS patients are at increased risk for infection, though infection rates have decreased significantly with better AIDS management . However, in spite of the reduction in AIDS-related cases, cryptococcosis remains a frequent life-threatening opportunistic mycosis for these patients in underdeveloped countries, and is a recently emergent disease in the United States Pacific Northwest  for as yet, unexplained reasons.
In this study we have identified a novel mechanism in C. neoformans that leads to the production of hyphae, with or without basidiospores, by haploid cells (monokaryotic fruiting). This mechanism appears to be dependent on the cell cycle and initiates from cells in G2 arrest. It occurs in the absence of α-α mating and/or endoduplication, thereby demonstrating that monokaryotic fruiting can occur asexually. Previous studies have shown that sexual reproduction can occur between cells of the same mating type, resulting in monokaryotic fruiting , and that this phenomenon occurs in nature . Under the specific conditions of this study, notably a 37°C seed culture temperature, we saw no evidence of an α-α cell fusion event, nor did we find evidence of endoduplication within the hyphae even though we screened hyphae that had produced basidiospores. During C. neoformans basidiosporogenesis, sexual reproduction results in meiosis in the basidium followed by successive mitotic divisions that yield the nuclei, which ultimately are inserted into spores as they form on the basidial surface . Lin et al. observed that when fruiting was derived from an α-α fusant, sporogenesis was robust with spore chains that were long and phenotypically similar to α-a mating during sexual reproduction . This process was found to be impaired in dmc1 mutants, which are meiotic mutants that still produce spores, but at a much lower frequency than sexually produced spores, and with truncated spore chains that sometimes occur as dyads (two rather than four chains) . The phenotype of basidiospores produced in the dmc1 strains was strikingly similar to what we observed in this study and what was previously reported . These observations may suggest that basidiosporogenesis can occur mitotically without meiosis, although we cannot exclude a duplication event in the basidium immediately followed by meiosis and sporogenesis. We did, however, test a dmc1 mutant and found that it was able to undergo monokaryotic fruiting under our conditions (data not shown).