The Cryptococcus neoformans STE12α gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)α cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12α. The ability to form hyphae, however, was not affected by deletion of STE12α when convergently growing MATa strains were present. Furthermore, ste12α disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12α disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12α locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12α cells than those of mice infected with STE12α cells. Using reporter gene constructs, we found that STE12α controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, CAP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III, which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.

Cryptococcus neoformans STE12␣, a homologue of Saccharomyces cerevisiae STE12, exists only in MAT␣ strains. We identified another STE12 homologue, STE12a, which is MATa specific. As in the case with ⌬ste12␣, the mating efficiency for ⌬ste12a was reduced significantly. The ⌬ste12a strains surprisingly still mated with ⌬ste12␣ strains. In MAT␣ strains, STE12a functionally complemented STE12␣ for mating efficacy, haploid fruiting, and regulation of capsule size in the mouse brain. Furthermore, when STE12a was replaced with two copies of STE12␣, the resulting MATa strain produced hyphae on filament agar. STE12a regulates mRNA levels of several genes that are important for virulence including CNLAC1 and CAP genes. STE12a also modulates enzyme activities of phospholipase and superoxide dismutase. Importantly, deletion of STE12a markedly reduced the virulence in mice, as is the case with STE12␣. Brain smears of mice infected with the ⌬ste12a strain showed yeast cells with a considerable reduction in capsule size compared with those infected with STE12a strains. When the disrupted locus of ste12a was replaced with a wild-type STE12a gene, both in vivo and in vitro mutant phenotypes were reversed. These results suggest that STE12a and STE12␣ have similar functions, and that the mating type of the cells influences the alleles to exert their biological effects. C. neoformans, thus, is the first fungal species that contains a mating-type-specific STE12 homologue in each mating type. Our results demonstrate that mating-typespecific genes are not only important for saprobic reproduction but also play an important role for survival of the organism in host tissue. C ryptococcus neoformans is a pathogenic fungus that primarily infects patients with impaired immune systems, but people with no known underlying immunodeficiencies are affected also (1). C. neoformans is a bipolar heterothallic species in which mating is controlled by two alleles: MAT␣ and MATa (2, 3). In the laboratory, the sexual reproduction cycle begins when two strains of the opposite mating type are crossed under nutrientdeprivation conditions. This cycle is characterized by the formation of dikaryotic hyphae with typical basidiomycetous clamp connections and by the production of basidia. Basidiospores germinate to produce yeast cells that multiply by polar budding without hyphal formation. Previous studies indicate that MAT␣ strains are found far more frequently than MATa strains among clinical as well as environmental isolates of serotype D (4). For serotype A strains, MAT␣ is thus far the only mating type recovered. In a mouse systemic-infection model, MAT␣ strains of serotype D are significantly more virulent than MATa strains, which suggests an important role of the MAT␣ locus for fungal pathogenicity (5). In the absence of MATa strains, MAT␣ strains also undergo haploid fruiting in response to nitrogen starvation, which results in hyphal production and sporulation (6).The signal-transduction pathways regulating morphogenesis and pathogenicity of C. neofor...