Abstract
Cryptococcus neoformans and C. gattii are pathogenic basidiomycetous yeasts and the commonest cause of fungal infection of the central nervous system. Cryptococci are typically haploid but several inter-species, inter-varietal and intra-varietal hybrids have been reported. It has a bipolar mating system with sexual reproduction occurring normally between two individuals with opposite mating types, α and a. This study set out to characterize hybrid isolates within the C. neoformans/C. gattii species complex: seven unisexual mating intra-varietal VNI/VNII (αAAα) and six novel inter-varietal VNII/VNIV (aADα). The URA5-RFLP pattern for VNII/VNIV (aADα) differs from the VNIII (αADa) hybrids. Analysis of the allelic patterns of selected genes for AD hybrids showed 79% or more heterozygosis for the studied loci except for CBS132 (VNIII), which showed 50% of heterozygosity. MALDI-TOF MS was applied to hybrids belonging to different sero/mating type allelic patterns. All hybrid isolates were identified as belonging to the same hybrid group with identification scores ranging between 2.101 to 2.634. All hybrids were virulent when tested in the Galleria mellonella (wax moth) model, except for VNII/VNIV (aADα) hybrids. VNI/VGII hybrids were the most virulent hybrids. Hybrids recovered from larvae manifested a significant increase in capsule and total cell size and produced a low proportion (5–10%) of giant cells compared with the haploid control strains. All strains expressed the major virulence factors—capsule, melanin and phospholipase B—and grew well at 37°C. The minimal inhibitory concentration of nine drugs was measured by micro-broth dilution and compared with published data on haploid strains. MICs were similar amongst hybrids and haploid parental strains. This is the first study reporting natural same sex αAAα intra-varietal VNI/VNII hybrids and aADα inter-varietal VNII/VNIV hybrids.
Highlights
The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are important human pathogens infecting mainly immunocompromised and immunocompetent individuals, respectively [1, 2]
The following strains were included as references for specific tests, e.g. mating type, serotype, virulence phenotype and pathogenicity studies: H99 (VNI, Aα) [39], IUM 96–2828 (VNI, Aa) [40], JEC21 (VNIV, Dα) [41], JEC20 (VNIV, Da) [41], CBS132 (VNIII, αADa) [11, 42], IUM 91–0804 (VNIII, aADα) [11], CDC R265 (VGIIa, Bα) [43] and CDC R272 (VGIIb, Bα) [44]
From the 365 isolates studied, 19 isolates were identified as hybrids including 4 inter-species isolates previously described [19], seven intra-varietal (VNI/VNII, αAAα) and eight inter-varietal (VNII/VNIV, aADα) hybrid isolates
Summary
The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are important human pathogens infecting mainly immunocompromised and immunocompetent individuals, respectively [1, 2]. Grubii (serotype A; molecular types VNI and VNII) and C. neoformans var. The sibling species C. gattii has four widely accepted molecular types (VGI, VGII, VGIII, VGIV) distributed amongst two serotypes (B and C) [4, 16]. Neoformans VNIV × C. gattii VGI), from HIV-negative patients in the Netherlands and one AB hybrid Grubii VNI × C. gattii VGI), from an HIV-positive patient in Canada [17, 18]. Grubii VNI × C. gattii VGII) from HIV-negative patients in Brazil and Colombia and one AB hybrid Grubii VNI × C. gattii VGI) from an HIV-positive patient in India have been described [19] Three AB hybrids (C. neoformans var. grubii VNI × C. gattii VGII) from HIV-negative patients in Brazil and Colombia and one AB hybrid (C. neoformans var. grubii VNI × C. gattii VGI) from an HIV-positive patient in India have been described [19]
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