The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein beta subunit, GPB1, was cloned and disrupted. gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATalpha and MATa cells. The functions of GPB1 are distinct from those of the Galpha protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence. gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show that MATa cells stimulate monokaryotic fruiting of MATalpha cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gbeta subunit GPB1 and the Galpha subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.

FIG. 1

C. neoformans GPB1 exhibits identity to G-protein β subunits. The sequences of Gβ subunits from humans (12), D. melanogaster (D.m.) (66), Cryphonectria parasitica (C.p.) (21), Schizosaccharomyces pombe (S.p.) (23), and S. cerevisiae (S.c.) (59) were aligned with that of the C. neoformans (C.n.) GPB1 protein. Identical amino acids are boxed and darkly shaded; conservative amino acid substitutions are boxed and lightly shaded.

FIG. 2

Disruption of the C. neoformans GPB1 gene. (A) A schematic illustration of the GPB1 gene replacement; (B) Southern analysis of the wild type and the gpb1 mutant. The ADE2 gene was inserted at an ApaI site in the GPB1 coding domain, and the gpb1::ADE2 disruption allele was used to biolistically transform the Δade2 strain M049 to adenine prototrophy. Genomic DNAs from the isogenic GPB1 wild-type strain H99 and the gpb1::ADE2 disruption mutant were isolated, cleaved with HindIII (H) or with NotI (N) and XbaI (X), separated by 1% agarose gel electrophoresis, transferred to a nylon membrane, and probed with the ³²P-labeled GPB1 open reading frame (indicated by an arrow labeled “probe”). Sizes of DNA fragments resulting from gene disruption are indicated by horizontal arrows. The positions of DNA molecular size standards are indicated on the left.

FIG. 3

The C. neoformans G-protein β subunit GPB1 is required for mating. The isogenic C. neoformans wild-type MATα strain H99 (GPB1 GPA1) and the gpb1::ADE2 (gpb1) and gpa1::ADE2 (gpa1) MATα mutant strains were mated with the MATa strain JEC20 on V8 agar medium (upper panels) and V8 agar medium supplemented with 2 or 10 mM cAMP as indicated (lower panels). The wild-type GPB1 gene was reintroduced into the gpb1 mutant strain as described in Materials and Methods (gpb1+GPB1). Mating was at 22°C for 7 days. Magnification, ×25.

FIG. 4

The Gα subunit GPA1 is not required for responses to pheromones. (A) Cells of the wild-type MATα serotype D strain JEC21 were grown in confrontation with the isogenic MATa GPA1 wild-type strain JEC20 (upper panel) or the gpa1 mutant strain BAC20 (lower panel), with incubation for 3 days at 24°C on filament agar, and conjugation tubes were photographed. Magnification, ×25. (B) A ura5 derivative of the GPA1 wild-type strain JEC20 (MATa ura5) and the isogenic gpa1 mutant strain BAC20 (MATa gpa1::ADE2 ura5) were transformed with plasmid pCnTel1 lacking or expressing the MFα1 pheromone gene, grown on filament agar for 2 days at 24°C, and photographed. Magnification, ×50.

FIG. 5

GPB1 is not required for virulence factors or virulence in C. neoformans. (A) The isogenic GPB1 GPA1 wild-type strain H99 and the gpb1::ADE2 (gpb1) and gpa1::ADE2 (gpa1) mutant strains were grown on niger seed agar for 72 h at 37°C. Strains that produce melanin (GPB1 GPA1, gpb1) form brown colonies on this medium, whereas strains that do not produce melanin (gpa1) are white. (B) Cells of the wild-type strain H99 (GPB1 GPA1) and the gpb1::ADE2 (gpb1) and gpa1::ADE2 (gpa1) mutant strains were grown in low-iron medium plus EDDHA at 30°C for 48 h to induce capsule synthesis. The polysaccharide capsule was identified by India ink staining and photographed. Magnification, ×200. (C) The GPB1 wild-type (H99) and gpb1 mutant strains were inoculated intracisternally into immunosuppressed rabbits. CSF was withdrawn on days 4, 7, 10, and 14 postinfection, and the numbers of surviving yeast cells were determined by plating serial dilutions of CSF on YPD medium. The mean cell count for each strain was plotted with the standard error of the mean.

FIG. 6

GPB1 activates a MAP kinase cascade involving the CPK1 kinase. (A) The CPK1 gene expressed from the C. neoformans GAL7 promoter in the URA5 plasmid pCnTel1 was introduced into a gpb1 ura5 mutant strain (see Materials and Methods) by biolistic transformation. The isogenic MATα wild-type strain H99 (GPB1 GPA1), the gpb1 mutant strain, and the gpb1 mutant strain transformed with the GAL7-CPK1 gene fusion (gpb1 GAL7-CPK1) were cocultured with a MATa mating partner (JEC20). Mating was for 21 days at 22°C on filament agar containing 0.5% galactose (shown here) or 0.5% glucose (data not shown). Magnification, ×25. (B) The congenic serotype D MATa ura5 strain JEC34 and the MATα ura5 strain JEC43 were transformed with the GAL7-GPB1 gene fusion linked to the URA5 gene and grown for 72 h at 24°C on filament agar with glucose or galactose. Conjugation tubes emanating from cell patches were photographed. Magnification, ×25.

FIG. 7

GPB1 and MATa cells regulate haploid fruiting. (A) The isogenic GPB1 wild-type strain H99 (far-left panel), the gpb1::ADE2 mutant strain (second panel from left), and the gpb1::ADE2 mutant strain reconstituted with the GPB1 wild-type gene (third panel from left) were transformed with the dominant active Ras1 Q67L mutant gene, grown on glucose filament agar medium for 7 days at 24°C, and photographed. The gpb1 mutant strain was also transformed with plasmid pCGS-1 expressing the GAL7-STE12 fusion gene and grown on galactose filament agar (far-right panel) for 7 days at 24°C. Magnification, ×25. (B) Cells of the serotype D MATα strain JEC21 were grown in confrontation with themselves (middle panel) or with congenic cells of the opposite (MATa) mating type (strain JEC20) (lower panel). As a control, the MATa strain JEC20 was grown in confrontation with itself (upper panel). Cells were incubated for 10 days at 24°C on filament agar and photographed. Magnification, ×25.