Variability of karyotypes and RAPD types in genetically related strains of Cryptococcus neoformans (original) (raw)
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Cryptococcus neoformans population includes hybrid strains homozygous at mating-type locus
FEMS Yeast Research, 2006
Recent attempts to characterise the hybrid strains of Cryptococcus neoformans have led to the identification of a cryptic population of hybrid strains ('H strains') with double DNA content but only a single mating-type allele. To verify a set of hypotheses concerning their origin, we investigated 14 previously isolated H strains and ten F1-progeny strains arising from H99 and JEC20 mating. The double DNA content was tested by flow cytometry; the presence of only one mating type was tested by amplifying 12 mating-type-specific genes and one gene unlinked with the mating-type locus (URA5). Analysis of the F1 progeny identified two H strains, and electrophoretic karyotyping confirmed the occurrence of genetic recombination. The simultaneous presence of the homozygous and heterozygous loci, and the fact that all of the F1-progeny strains presented a recombinant karyotype, suggest that the H strains originated from the post-meiotic random fusion of two of the four recombinant nuclei. Further studies are required to elucidate the role of the homozygous mating-type loci in the virulence of C. neoformans.
Pathogens
Hybrids between Cryptococcus neoformans and Cryptococcus deneoformans are commonly found in patients and the environment. However, the genetic stability of these hybrids remains largely unknown. Here, we established mutation accumulation lines of a diploid C. neoformans × C. deneoformans laboratory hybrid and analyzed the genotypes at 33 markers distributed across all 14 chromosomes. Our analyses found that under standard culture conditions, heterozygosity at most loci was maintained over 800 mitotic generations, with an estimated 6.44 × 10−5 loss-of-heterozygosity (LoH) event per mitotic division. However, under fluconazole stress, the observed LoH frequency increased by > 50 folds for the two markers on Chromosome 1, all due to the loss of the fluconazole susceptible allele on this chromosome. Flow cytometry analyses showed that after the 40th transfer (120 days), 19 of the 20 lines maintained the original ploidy level (2N), while one line was between 2N and 3N. The combined fl...
Infection and Immunity, 2001
Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1 , CLA4 , and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MAT a or MAT α mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MAT α locus was derived from the serotype D parent and the MAT a locus was inherited from a serotype A parent that h...
F1000 - Post-publication peer review of the biomedical literature, 2009
The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the a mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual a population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (a-a) also occurs in nature as evidenced by the existence of natural diploid aADa hybrids that arose by fusion between two a cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ,500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the a MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct a cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.
Chromosome length polymorphism in Cryptococcus neoformans clinical and environmental isolates
Revista iberoamericana de micología, 2001
A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C. neoformans isolates displaying different degrees of capsule formation. A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C. neoformans var. neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C. neoformans var. neoformans and 13 isolates of C. neoformans var. gattii. Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii. The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains). No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespect...
Phenotypic Switching and Genetic Diversity of Cryptococcus neoformans
Journal of Clinical Microbiology, 2001
Niger seed agar was used as a primary plating medium for the isolation of Cryptococcus neoformans from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans, C. neoformans may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiplestrain infections.
Memórias do Instituto Oswaldo Cruz, 2007
Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance -that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture.
Extensive allelic variation in Cryptococcus neoformans
Journal of clinical microbiology, 1992
The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C....
PLoS Pathogens, 2009
The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the a mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual a population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (a-a) also occurs in nature as evidenced by the existence of natural diploid aADa hybrids that arose by fusion between two a cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ,500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the a MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct a cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.