Origin of Cryptococcus neoformans var. neoformans diploid strains

The basidiomycetous yeast Cryptococcus neoformans is an important human fun gal pathogen. Two varieties, C. neoformans var. neoformans and C. neoforman s var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MAT alpha Some rare isolates are self-fertile and are cons idered occasional diploid or aneuploid strains. In the present study, 133 i solates, mostly from Italian patients, were investigated to detect the pres ence of diploid strains in the Igiene University Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that t he diploid state was associated with two specific genotypes identified as V N3 and VN4. Determination of mating type on VS juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the tw o pheromone genes (MFa and MF alpha) as probes identified 36 of the 49 dipl oid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFa genes exist and are distinct for serotypes A and D. I n contrast, the MFa gene sequence was conserved in both serotype alleles. A mplification of serotype-specific STE20 alleles demonstrated that the diplo id strains contained one mating locus inherited from a serotype A parent an d one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.