Cryptococcus neoformans is a globally distributed human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. It has a defined sexual cycle involving haploid cells of alpha and a mating types, yet the vast majority of environmental and clinical isolates are alpha (ref. 3). Sexual recombination is normally expected to occur between isolates of opposite mating type in organisms with two mating types (or sexes). How sexual reproductive potential can be maintained in an organism with a largely unisexual, nearly clonal population genetic structure is unknown. One clue, however, is that alpha strains undergo fruiting, a process that resembles sexual mating but is thought to be strictly mitotic and asexual. We report here that hallmarks of mating occur during fruiting, including diploidization and meiosis. Pheromone response pathway elements and the key meiotic regulator Dmc1 are required for efficient fruiting. Furthermore, fusion and meiosis can occur between non-isogenic alpha strains, enabling genetic exchange. These studies reveal how sexual reproduction can occur between partners of the same mating type. These findings have implications for the evolution of microbial pathogens, as well as for parthenogenesis, cell fusion events and transitions between self-fertilizing and outcrossing modes of reproduction observed in both fungi and other kingdoms.

Since its classification nearly 80 years ago, the human pathogen Candida albicans has been designated as an asexual yeast. In this report, we describe the construction of C. albicans strains that were subtly altered at the mating-type-like (MTL) locus, a cluster of genes that resembles the mating-type loci of other fungi. These derivatives were capable of mating after inoculation into a mammalian host. C. albicans is a diploid organism, but most of the mating products isolated from a mouse host were tetrasomic for the two chromosomes that could be rigorously monitored and, overall, exhibited substantially higher than 2n DNA content. These observations demonstrated that C. albicans can recombine sexually.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Candida albicans, the most prevalent fungal pathogen in humans, is thought to lack a sexual cycle. A set of C. albicans genes has been identified that corresponds to the master sexual cycle regulators a1, alpha1, and alpha2 of the Saccharomyces cerevisiae mating-type (MAT) locus. The C. albicans genes are arranged in a way that suggests that these genes are part of a mating type-like locus that is similar to the mating-type loci of other fungi. In addition to the transcriptional regulators a1, alpha1, and alpha2, the C. albicans mating type-like locus contains several genes not seen in other fungal MAT loci, including those encoding proteins similar to poly(A) polymerases, oxysterol binding proteins, and phosphatidylinositol kinases.

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal ␤-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

Cryptococcus neoformans is a pathogenic fungus that primarily afflicts immunocompromised patients, infecting the central nervous system to cause meningoencephalitis that is uniformly fatal if untreated. C. neoformans is a basidiomycetous fungus with a defined sexual cycle that has been linked to differentiation and virulence. Recent advances in classical and molecular genetic approaches have allowed molecular descriptions of the pathways that control cell type and virulence. An ongoing genome sequencing project promises to reveal much about the evolution of this human fungal pathogen into three distinct varieties or species. C. neoformans shares features with both model ascomycetous yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe) and basidiomycetous pathogens and mushrooms (Ustilago maydis, Coprinus cinereus, Schizophyllum commune), yet ongoing studies reveal unique features associated with virulence and the arrangement of the mating type locus. These advances have catapulted C. neoformans to center stage as a model of both fungal pathogenesis and the interesting approaches to life that the kingdom of fungi has adopted.

Candida lusitaniae is an emerging human pathogen that, unlike other fungal pathogens, frequently develops resistance to the commonly used antifungal agent amphotericin B. Amphotericin B is a member of the polyene class of antifungal drugs, which impair fungal cell membrane integrity. Here we analyzed mechanisms contributing to amphotericin B resistance in C. lusitaniae. Sensitivity to polyenes in the related fungi Saccharomyces cerevisiae and Candida albicans requires the ergosterol biosynthetic gene ERG6. In an effort to understand the mechanisms contributing to amphotericin B resistance in C. lusitaniae, we isolated the ERG6 gene and created a C. lusitaniae erg6⌬ strain. This mutant strain exhibited a growth defect, was resistant to amphotericin B, and was hypersensitive to other sterol inhibitors. Based on the similarities between the phenotypes of the erg6⌬ mutant and clinical isolates of C. lusitaniae resistant to amphotericin B, we analyzed ERG6 expression levels and ergosterol content in multiple clinical isolates. C. lusitaniae amphotericin B-resistant isolates were found to have increased levels of ERG6 transcript as well as reduced ergosterol content. These changes suggest that another gene in the ergosterol biosynthetic pathway could be mutated or misregulated. Further transcript analysis showed that expression of the ERG3 gene, which encodes C-5 sterol desaturase, was reduced in two amphotericin B-resistant isolates. Our findings reveal that mutation or altered expression of ergosterol biosynthetic genes can result in resistance to amphotericin B in C. lusitaniae.

Homeodomain proteins are central regulators of development in eukaryotes. In fungi, homeodomain proteins have been shown to control cell identity and sexual development. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle that produces spores, the suspected infectious particles. Previously, only a single homeodomain regulatory protein involved in sexual development, Sxi1␣, had been identified. Here we present the discovery of Sxi2a, a predicted but heretofore elusive cell-type-specific homeodomain protein essential for the regulation of sexual development. Our studies reveal that Sxi2a is necessary for proper sexual development and sufficient to drive this development in otherwise haploid ␣ cells. We further show that Sxi1␣ and Sxi2a interact with one another and impart similar expression patterns for two key mating genes. The discovery of Sxi2a and its relationship with Sxi1␣ leads to a new model for how the sexual cycle is controlled in C. neoformans, with implications for virulence.Patterning during development in animals is controlled by a special set of coordinately regulated genes, the homeobox genes. These genes encode homeodomain proteins that act in concert with other cellular factors to establish patterns in diverse organisms. For example, in the fruit fly Drosophila melanogaster, where homeobox genes were first discovered, expression of homeodomain proteins specifies identity along the anteroposterior axis (17). In plants, homeodomain proteins regulate, among other things, the patterning of petals in flowers (25). These proteins are also important in controlling sexual differentiation, as has been documented in the mouse (45).