Determination of ploidy in<i>Cryptococcus neoformans</i>by flow cytometry

Tanaka, Reiko; Taguchi, Hideaki; Takeo, Kanji; Miyaji, Makoto; Nishimura, Kazuko

doi:10.1080/02681219680000521

Cited by 54 publications

(46 citation statements)

References 10 publications

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“…The fluorescence-activated cell-sorting (FACS) protocol was modified from Tanaka et al (15). Cells were grown overnight at 25°C in yeast extract-peptone-dextrose (YPD) broth, collected by centrifugation, and washed with 1ϫ phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species

et al. 2013

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b Kwoniella mangrovensis has been described as a sexual species with a bipolar mating system. Phylogenetic analysis of multiple genes places this species together with Kwoniella heveanensis in the Kwoniella clade, a sister clade to that containing two pathogenic species of global importance, Cryptococcus neoformans and Cryptococcus gattii, within the Tremellales. Recent studies defining the mating type loci (MAT) of species in these clades showed that, with the exception of C. neoformans and C. gattii, which are bipolar with a single biallelic multigene MAT locus, several other species feature a tetrapolar mating system with two unlinked loci (homeodomain [HD] and pheromone/receptor [P/R] loci). We characterized several strains from the original study describing K. mangrovensis; two MAT regions were amplified and sequenced: the STE20 gene (P/R locus) and the divergently transcribed SXI1 and SXI2 genes (HD locus). We identified five different mating types with different STE20/SXI allele combinations that together with results of mating experiments demonstrate that K. mangrovensis is not bipolar but instead has a tetrapolar mating system. Sequence and gene analysis for a 43-kb segment of the K. mangrovensis type strain MAT locus revealed remarkable synteny with the homologous K. heveanensis MAT P/R region, providing new insights into slower evolution of MAT loci in the Kwoniella compared to the Cryptococcus clade of the Tremellales. The study of additional isolates from plant substrates in Europe and Botswana using a combination of multilocus sequencing with MAT gene analysis revealed two novel sibling species that we name Kwoniella europaea and Kwoniella botswanensis and which appear to also have tetrapolar mating systems. Fungal mating-type loci (MAT) are specialized regions of the genome that determine sexual identity of haploid cells and progression through the sexual cycle (1). There is considerable interest in the genetic characterization of MAT loci due to their central role in fungal life cycles, their connection to lifestyle and virulence (viz., in human or plant-pathogenic taxa), and the impact of sexual recombination on population genetics and speciation. The discovery of remarkable convergence in the structure of sex-determining genomic regions from studies in animals, plants, and fungi illuminates the forces that shape the evolutionary trajectories of MAT loci or sex chromosomes in eukaryotes (2).In basidiomycetes, two major types of MAT loci have so far been recognized (3). The tetrapolar mating system of the corn smut Ustilago maydis or the mushroom Coprinopsis cinerea is governed by two small (Ͻ10-kb) unlinked loci: one encodes members of the homeodomain (HD) family of transcription factors, which heterodimerize upon mating to generate an active transcription regulator (HD locus), and the other encodes lipopeptide pheromone precursors and 7-transmembrane pheromone receptors that mediate intercellular signaling (P/R locus). Alleles at both loci must differ for mating to occur, and in many cases each...

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Section: Methodsmentioning

confidence: 99%

Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species

et al. 2013

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“…Ploidy was determined for each isolate by measuring cellular DNA content, using a modified protocol for flow cytometry (36,63). Control strains were the haploid H99 strain and our AD hybrid diploid strain RSA5272 (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Analyses of Pediatric Isolates of Cryptococcus neoformans from South Africa

et al. 2011

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Compared to the incidence in adults, cryptococcosis is inexplicably rare among children, even in subSaharan Africa, which has the highest prevalence of coinfection with HIV and Cryptococcus neoformans. To explore any mycological basis for this age-related difference in the incidence of cryptococcosis, we investigated isolates of C. neoformans recovered from pediatric and adult patients during a 2-year period in South Africa. From reports to the Group for Enteric, Respiratory, and Meningeal Disease Surveillance in South Africa (GERMS-SA), we reviewed all cases of cryptococcosis in 2005 and 2006. We analyzed one isolate of C. neoformans from each of 82 pediatric patients (<15 years of age) and determined the multilocus sequence type (ST), mating type, ploidy, and allelic profile. This sample included isolates of all three molecular types of serotype A or C. neoformans var. grubii (molecular types VNI, VNII, and VNB) and one AD hybrid. Seventyseven (94%) of the strains possessed the MAT␣ mating type allele, and five were MATa. Seventy-five (91%) were haploid, and seven were diploid. A total of 24 different STs were identified. The ratios of each mating type and the proportion of haploids were comparable to those for the isolates that were obtained from 86 adult patients during the same period. Notably, the most prevalent pediatric ST was significantly associated with male patients. Overall, these pediatric isolates exhibited high genotypic diversity. They included a relatively large percentage of diploids and the rarely reported MATa mating type.

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“…Fluorescence-activated cell sorter (FACS) analysis was performed as described previously by Tanaka et al (40). Ploidy was determined in a selection of fertile and nonfertile isolates by comparison to previously characterized control haploid and diploid strains.…”

Section: Methodsmentioning

confidence: 99%

Clinical and Environmental Isolates of Cryptococcus gattii from Australia That Retain Sexual Fecundity

et al. 2005

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Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor ϳ40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MAT␣-specific SXI1␣ gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MAT␣ strains sequenced, the SXI1␣ gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.

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Determination of ploidy inCryptococcus neoformansby flow cytometry

Cited by 54 publications

References 10 publications

Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species

Molecular and Genetic Evidence for a Tetrapolar Mating System in the Basidiomycetous Yeast Kwoniella mangrovensis and Two Novel Sibling Species

Analyses of Pediatric Isolates of Cryptococcus neoformans from South Africa

Clinical and Environmental Isolates of Cryptococcus gattii from Australia That Retain Sexual Fecundity

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