Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans

Abstract

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

FIG. 1.

Disruption of the adenylyl cyclase gene CAC1 abolishes cAMP production. (A) CAC1 wild-type (WT) (H99), cac1 mutant (RPC3), cac1 CAC1-reconstituted (RPC7), and gpa1 CAC1 (AAC17) strains were incubated in YPD medium for 18 h. The cells were divided for incubation for 4 h in one of three different media: synthetic complete medium with 2% glucose (nitrogen +, glucose +), synthetic complete medium with 0% glucose (nitrogen +, glucose −), or SLAD (nitrogen −, glucose +). Total RNA from each sample was assessed by Northern analysis with the CAC1 gene as a probe. The rRNA bands of the ethidium bromide-stained gel (rRNA) are shown to demonstrate RNA loading. (B) CAC1 wild-type (H99) (squares), cac1 mutant (LCC22) (diamonds), cac1 CAC1-reconstituted (LCC22-1) (triangles), gpa1 mutant (AAC1) (circles), gpa1 GPA1-reconstituted (AAC3) (crossed squares), and gpa1 CAC1 (AAC17) (crosses) strains were starved for glucose for 2 h. At the indicated time after a glucose pulse, aliquots of the cell suspensions were frozen, and intracellular cAMP concentrations were determined. Data points represent the mean and standard deviation for duplicate samples in two identical experiments (four samples for each data point).

FIG. 2.

Adenylyl cyclase is required for mating in C. neoformans. (A) CAC1 wild-type, cac1 mutant, cac1 CAC1-reconstituted, gpa1 mutant, and gpa1 CAC1 strains were coincubated with the MATa strain JEC20 on V8 mating medium in the dark for 14 days at 30°C with and without cAMP (2.5 mM). (B) The edges of the mating mixtures were examined microscopically each day for mating hyphae and photographed after 7 days (×61).

FIG. 3.

Adenylyl cyclase mutants have defects in melanin production. CAC1 wild-type (WT), cac1 mutant, cac1 CAC1-reconstituted, gpa1 mutant, and gpa1 CAC1 strains were grown on Niger seed medium with (+) and without (−) cAMP (2.5 mM) at 37°C and photographed after 4 days. Strains that produce melanin are brown (gray on figure), whereas strains that produce less or no melanin are white.

FIG. 4.

Adenylyl cyclase mutants fail to induce capsule. (A) CAC1 wild-type (WT), cac1 mutant, cac1 CAC1-reconstituted, gpa1 mutant, and gpa1 CAC1 strains were incubated for 2 days under capsule-inducing conditions (DMEM–22 mM NaHCO₃) with and without cAMP (20 mM). Capsule induction was qualitatively assessed with a standard India ink preparation and photographed (×61). (B) Capsule size was quantified by determining the packed-cell volume of normalized cell suspensions (10⁹ cells/ml) for each sample. Data points represent the mean and standard error for triplicate samples.

FIG. 5.

Adenylyl cyclase is required for virulence of C. neoformans. Ten A/Jcr mice were intranasally inoculated with the CAC1 wild-type strain (diamonds), the cac1 mutant strain (squares), or the cac1 CAC1-reconstituted strain (triangles). The three groups of mice (30 total) were monitored for survival over 60 days.