Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a pathogenic fungus with a defined sexual cycle involving haploid MATalpha and MATa cells. Interestingly, MATalpha strains are more common, are more virulent than congenic MATa strains, and undergo haploid fruiting in response to nitrogen limitation or MATa cells. Three genes encoding the MFalpha pheromone were identified in the MATalpha mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with MATa cells. The MFalpha1, MFalpha2, and MFalpha3 genes were mutated, individually and in combination. MATalpha strains lacking MFalpha pheromone failed to induce morphological changes in MATa cells. Pheromoneless MATalpha mutants were fusion and mating impaired but not sterile and mated at approximately 1% the wild-type level. The pheromoneless MATalpha mutants were also partially defective in haploid fruiting, and overexpression of MFalpha pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of MATalpha cells and stimulated conjugation tube formation in MATa cells. A conserved G-protein activated mitogen-activated protein kinase signaling pathway was found to be required for both induction and response to mating pheromones. The MFalpha pheromone was not essential for virulence of C. neoformans but does contribute to the overall virulence composite. These studies define paracrine and autocrine pheromone response pathways that signal mating and differentiation of this pathogenic fungus.

FIG. 1.

Disruption and characterization of the MFα pheromone genes. (A) The disruption alleles of the MFα1 and MFα2,3 genes were constructed as described in Materials and Methods. Genomic DNA was isolated from the MFα1,2,3 wild-type strain, one mfα1Δ::ADE2 mutant strain, four mfα2,3Δ::URA5 mutant strains, and five mfα1Δ::ADE2 mfα2,3Δ::URA5 mutant strains; digested with XhoI and HindIII; electrophoresed; and analyzed by Southern hybridization with the common open reading frame of the MFα genes as a probe. The GPA2 gene encoding a C. neoformans heterotrimeric G-protein α subunit was used as a control. (B) Total RNA was isolated from the MATα wild-type, mfα1, mfα2,3, and mfα1,2,3 strains grown on V8 agar medium in the presence or absence of the wild-type MATa strain JEC20. Then, 10 μg of total RNA was analyzed with the MFα common, MFα3 specific, and actin gene probes. (C) Total RNA was isolated from MATα lys1 (JEC31), MATα ura5 (JEC43), and MATα ade2 (JEC50) auxotrophic strains grown on V8 agar medium or V8 supplemented with 30 μg of lysine, 20 μg of uracil, or 20 μg of adenine/ml, respectively. Next, 10 μg of total RNA was analyzed with the MFα common and actin gene probes.

FIG. 2.

MFα pheromone promotes but is not essential for mating. The isogenic wild-type (JEC21), mfα1,2,3 (WSC18), and mfα1,2,3 plus MFα1 (WSC56) strains were coincubated with a MATa wild-type strain (JEC20) on V8 agar mating medium in room light for 2 days at 24°C. The edges of the mating mixtures were photographed at ×100 magnification.

FIG. 3.

MFα pheromone regulates morphogenesis of confronting MATa cells. The MATα wild-type (JEC21), mfα1,2,3 (WSC18), and MFα1,2,3 MFα1 (WSC56) strains were incubated in close proximity in confrontation assays with the MATa strain JEC20 on filament agar and photographed at ×100 magnification (left panels) or at ×200 magnification after 120 h incubation at 24°C (right panels).

FIG. 4.

MFα pheromone stimulates haploid fruiting of C. neoformans. (A) The isogenic wild-type (JEC21), mfα1,2,3 (WSC18), and mfα1,2,3 plus MFα 1 (WSC56) strains were incubated on filament agar for 10 days at 24°C in the dark. (B) The isogenic wild type (JEC21), the mfα1,2,3 mutant strain (WSC17), the MATα ura5 wild-type strain JEC43 transformed with the P_GPD1-MFα1 gene fusion, and the mfα1,2,3 ura5 mutant strain (WSC50) transformed with the P_GPD1-MFα1 gene fusion (strain WSC57) were incubated on filament agar for 10 days at 24°C in the dark. The edges of the colony were photographed at ×100 magnification.

FIG. 5.

Pheromones signal haploid fruiting and mating associated gene activation via a conserved G-protein-activated MAP kinase pathway. (A) MFα and MFa pheromones enhance haploid fruiting of wild-type cells but not gpb1 or ste7 mutant cells. The isogenic MATα ura5 wild-type strain (JEC43) and the mfα1,2,3 (WSC50), gpb1 (WSC131), and ste7 (RDC21) mutant strains were biolistically transformed with the MFα1 (pRCD17) or MFa1 (pRCD101) expression plasmids as described in Materials and Methods. Transformants were incubated on filament agar for 5 days at room temperature and photographed at ×100 magnification. (B) The Ste7 MAP kinase is required for activation of MFα pheromone gene expression by MATa cells. The congenic MATα wild-type strain JEC43 and the ste7 mutant strain RDC21 were transformed with the MFα1-lacZ reporter plasmid pRCD41 (18). Transformants were grown on V8 mating medium for 24 h, and the β-galactosidase activity was measured as described in Materials and Methods and plotted here in Miller units. (C) Both MFa1 and MFα1 pheromones stimulate conjugation tube formation by MATa cells. The MATa ura5 strain JEC34 was transformed with the MFa1 (pRCD101) or the MFα1 (pRCD17) pheromone expression plasmid by biolistics as described in Materials and Methods, and Ura⁺ transformants were incubated on filament agar for 2 days at room temperature and photographed at ×100 magnification.

FIG. 6.

The MFα pheromone is not essential for virulence. A total of 10⁷ cells of the wild-type strain (JEC21), the mfα1,2,3 mutant strain (WSC18), and two independent mfα1,2,3 meiotic segregants isolated after a genetic cross (strains RDC46-3 and RDC46-4) were each injected intravenously into groups of 10 C5 complement-deficient DBA mice. Survival was monitored for 40 days and plotted. Symbols: ◊, MATα; □, mfα1,2,3; ▵, mfα1,2,3-1; ○, mfα1,2,3-2.