Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates

Abstract

Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and alpha mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the alpha mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and alpha mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and alpha mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the alpha mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence.

FIG. 1.

Identification of a and α haploid serotype A C. neoformans strains. (A) FACS analysis of the aA strains 125.91, KNA13, and KNA14; the αA strain 8-1; and the control aD haploid strain JEC20 and aDDa diploid strain CHY600. (B) Southern blots of 125.91 (aA), 8-1 (αA), H99 (αA), JEC20 (aD), and JEC21 (αD) PstI-digested genomic DNAs probed with transposon T1 or transposon T2. (C) Structures of the MAT locus alleles from the aD strain JEC20, the aA strain 125.91, and the αA strain H99. The mating type-specific regions are shown as thick lines, and the flanking regions are shown as thin lines. Genes are represented as arrows in the direction of transcription. Genes encoding pheromones are shown as black arrows, locus-specific genes are shown as white arrows, and all other genes are in grey. The IKS1 genes are circled with a black line, the RPO41 genes are circled with a dotted line, and the NCP1a gene is boxed. (D) PCR assays were conducted with primers specific to NCP1a. Genomic DNAs from serotype D, serotype A, and AD hybrid strains were assayed. aADα hybrid strains were ZG290, ATCC 48184, CDC92-74 (ADα), CDC228, and CDC304. αADa hybrid strains were ZG287, MMRL774, KW5, CBS132, and CDC94-383. PCR with primers specific to the actin gene served as a positive control for the presence of genomic DNA.

FIG. 2.

Morphological features of C. neoformans var. grubii mating. The a mating type, serotype A strain 125.91 was mixed with the α mating type, serotype A strain 8-1. (A) Example of filaments observed after 10 days of growth on V8 (pH 5.0) at 25°C in the dark. Bar, 1 mm. (B) Example of filaments, basidia, and spores produced after 10 days of growth on V8 (pH 5.0) at 25°C in the dark. Bar, 25 μm. (C) DAPI staining of nuclei in filaments produced in a 125.91 (aA)-to-8-1 (αA) cross. Arrows designate paired nuclei in the dikaryons. Bar, 25 μm. (D) Differential contrast image of the filaments seen in panel C. (E) Calcofluor White staining of the septa of filaments produced in a 125.91 (aA)-to-8-1 (αA) cross. The arrowhead designates a fused clamp connection. Bar, 25 μm. (F) DIC image of the filament seen in panel E.

FIG. 3.

RFLP analysis of progeny from C. neoformans var. grubii crosses shows recombination. The presence or absence of the PvuI restriction site in the GPA1 gene and the BstXI restriction site in the PAK1 gene was examined by digestion of the PCR-amplified gene with the appropriate enzyme. An asterisk indicates the strain used to generate the serotype A congenic strains. (A) RFLP analysis of the parental strains 125.91 (aA) and 8-1 (αA) and progeny from a cross of the parental strains. Strain KNA14 was used to generate the serotype A congenic strains. (B) RFLP analysis of the parental strains KNA14 (aA) and H99 crg1 (αA) and progeny from a cross of the parental strains. KNB-5 was used to generate the serotype A congenic strains. (C) RFLP analysis of the parental strains KNA14 (aA) and H99 (αA), the 5th-backcross strain KN99-5a, and the 10th-backcross strains KN99a and KN99α.

FIG. 4.

The crg1 mutation enhances filamentation during mating of serotype A strains. The aA strain KNA14 was mated with the αA strain H99 or an isogenic crg1 αA mutant strain on V8 (pH 5.0) medium at 25°C in the dark for 10 days. (A) Example of aA (KNA14)-to-αA (H99) mating. Bar, 500 μm. (B) Filaments, basidia, and spores produced during an aA (KNA14)-to-αA (H99) mating. Bar, 50 μm. (C) Example of an aA (KNA14)-to-αA crg1 (H99 crg1) mutant mating. Bar, 100 μm. (D) Filaments, basidia, and spores produced during an aA (KNA14)-to-αA crg1 (H99 crg1) mutant mating. Bar, 50 μm.

FIG. 5.

Karyotype analysis of C. neoformans var. neoformans strains. Chromosomes from the aA strains 125.91, KNA14, KN99-5a, and KN99a and the αA strains 8-1, H99, and KN99α were separated by PFGE, and the gel was stained with ethidium bromide. Size markers are based on S. cerevisiae chromosomes.

FIG. 6.

The crg1 mutation enhances pheromone response. The a and α strains were grown in confronting lines ∼1 mm apart on SLAD medium and incubated at 25°C in the dark for 72 h. Strains were examined microscopically for the presence of conjugation tubes. aD, JEC20; αD, JEC21; αA, H99; αA crg1, H99 crg1. Bar, 1 mm.

FIG. 7.

Schematic diagram of the mating scheme used to produce a serotype A congenic strain pair. KN99-5a is the product of the 5th backcross and KN99a and KN99α are products of the 10th backcross to H99.

FIG. 8.

Virulence of serotype A strains. (A) Groups of 10 A/Jcr mice were infected with 10⁵ cells of the αA strains H99 and 8-1, the aA strain KNA14, and the fifth-backcross progeny KN99-5a (aA), and survival was monitored. (B) Groups of 10 A/Jcr mice were infected with 5 × 10⁴ cells of H99 (αA) and KN99-5a (aA), and survival was monitored. (C) Groups of three immunosuppressed rabbits were inoculated with 10⁸ cells of strains KN99a (aA), KN99α (αA), and JEC21 (αD). CSF was withdrawn on days 4 and 7 postinfection, and the number of surviving yeast cells was determined by plating serially diluted CSF on YPD medium. (D) Groups of 10 A/Jcr mice were infected with either 10⁵ or 10³ cells of strains H99 (αA), KN99a (aA), and KN99α (αA), and survival was monitored. Error bars indicate standard errors of the means.