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 Gα 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 Gα protein Gpa1,
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
gpa1
mutant strains. Physiological studies revealed that the Gα 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 Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.