Cryptococcus neoformans mating and virulence are regulated by the G-protein alpha subunit GPA1 and cAMP

Abstract

This study explores signal transduction pathways that function during mating and infection in the opportunistic, human fungal pathogen Cryptococcus neoformans. The gene encoding a G-protein alpha subunit homolog, GPA1, was disrupted by homologous recombination. The gpa1 mutant strain was viable but exhibited a defect in mating in response to nitrogen starvation. Additionally, the gpa1 mutant strain failed to induce two well-established virulence factors-melanin synthesis, in response to glucose starvation; and capsule production, in response to iron limitation. As a consequence, virulence of the gpa1 mutant strain was significantly attenuated in an animal model of cryptococcal meningitis. Reintroduction of the wild-type GPA1 gene complemented the gpa1 mutant phenotypes and restored mating, melanin and capsule production, and virulence. Similarly, exogenous cAMP also suppressed the gpa1 mutant phenotypes, restoring mating and production of melanin and capsule. These observations support a model in which GPA1 has a role in sensing diverse environmental signals required for mating and virulence by regulating cAMP metabolism in C. neoformans.

Figure 1

Disruption of the C. neoformans GPA1 gene. (A) The C. neoformans ADE2 gene was inserted into an NdeI site in exon III of the GPA1 gene to yield the gpa1::ADE2 disruption allele. The positions of primers 1 and 2 (see Materials and Methods) are indicated by arrows and the start (ATG) and stop (TAA TGA) codons of the GPA1 gene depicted. (B) Genomic DNA was isolated from the GPA1 wild-type strain, two gpa1::ADE2 mutant strains, and one strain in which the transformed gpa1::ADE2 allele had integrated only ectopically, digested with HindIII, electrophoresed, and analyzed by Southern hybridization with the GPA1 gene as probe. (C) Genomic DNA from the GPA1, gpa1, and gpa1 + GPA1 strains was amplified by PCR with GPA1-specific primers (1 and 2), electrophoresed in a 1% agarose gel, and stained with ethidium bromide. The open arrowhead indicates the gpa1::ADE2 allele and the solid arrow the wild-type GPA1 gene. (D) Total RNA was isolated from the GPA1 wild-type, the gpa1 mutant, the gpa1 mutant grown in nitrogen-deprivation media, and the gpa1 + GPA1 reconstituted strain. RNA was electrophoresed and analyzed by Northern hybridization with the GPA1 gene as probe. Before RNA transfer, the gel was stained with ethidium bromide to determine relative ribosomal RNA concentrations for each sample.

Figure 2

GPA1 regulates mating in C. neoformans. The isogenic GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 MATα strains were co-incubated with a MATa mating partner (strain JEC20) on nitrogen limiting mating media (V8 agar) for 7 days at 25°C. The edges of the mating mixtures were photographed (50×).

Figure 3

GPA1 regulates capsule formation in C. neoformans. The isogenic GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 strains were cultured for 48 hr at 30°C in liquid low iron media with 56 μm EDDHA (an iron chelator). The polysaccharide capsule was stained by a standard India ink preparation, and the cells were photographed (200×).

Figure 4

GPA1 regulates melanin production in C. neoformans. (A) The GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 strains were grown on Niger seed agar for 7 days at 37°C. Strains that produce melanin (GPA1, gpa1 + GPA1) are brown on this media, whereas strains that do not produce melanin (gpa1 mutant) are white. (B) The GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 strains were grown for 16 hr at 37°C with glucose starvation (as described in Materials and Methods). Cells were permeabilized with toluene:ethanol, incubated for 2 hr in the presence of the diphenolic substrate caffeic acid, and cellular phenoloxidase activity assayed spectrophotometrically by measuring the appearance of melanin in the supernatant by the change in absorbance at 480 nm. Values represent the mean of three separate but identical cultures for each strain; error bars indicate standard error of the mean.

Figure 5

GPA1 is required for virulence of C. neoformans in a rabbit model of cryptococcal meningitis. (A) Rabbits (four for each strain) were immunosuppressed with corticosteroids and inoculated intrathecally with 10⁸ cells of the isogenic GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 strains. CSF was removed on days 7, 10, and 14 following inoculation, and the number of surviving organisms was determined by serial dilution and plating on YPD medium. Each data point represents the mean of all cultures for each strain, and the standard error of the mean is indicated. (B) Equal volumes (100 μl) of CSF obtained from a rabbit infected with the GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 reconstituted strains were plated on YPD medium and incubated for 72 hr at 30°C.

Figure 6

Exogenous cAMP suppresses the gpa1 mutant mating defect. The GPA1 wild-type strain and gpa1 mutant strain were cocultured with a MATa mating partner (JEC20) on V8 mating media, without (−cAMP) and with (+cAMP) 10 mm cAMP. Following 7 days incubation at 25°C, the edges of the mating mixtures were photographed (50×).

Figure 7

Exogenous cAMP restores capsule and melanin production in the gpa1 mutant strain. (A) The GPA1 wild-type and gpa1 mutant strains were cultured for 24 hr in liquid low iron media (LIM + EDDHA), with and without 10 mm cAMP, and cells were examined with a standard India ink preparation and photographed (200×). (B) Cultures of the GPA1 wild-type and gpa1 mutant strains were suspended and normalized to 10⁸ cells/ml, and packed cell volumes were measured. Results presented are the mean of duplicate measurements of two separate but identical cultures, with standard error indicated. (C) The GPA1 wild-type, gpa1 mutant, and gpa1 + GPA1 strains were cultured for 7 days at 37°C on Niger seed agar, with and without 10 mm cAMP.

Figure 8

A model for the role of GPA1 in mating and virulence in C. neoformans. Our findings support a model in which the Gα protein GPA1 and cAMP transduce environmental signals that regulate mating and the induction of virulence factors (melanin and capsule) required for pathogenicity of C. neoformans. Other signals (e.g., pheromone) and signaling cascades (not shown) also likely operate in regulating differentiation and the acquisition of virulence.