Adenylyl Cyclase-Associated Protein Aca1 Regulates Virulence and Differentiation of Cryptococcus neoformans via the Cyclic AMP-Protein Kinase A Cascade

Abstract

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the G␣ subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.Cryptococcus neoformans is a heterothallic, basidiomycetous, pathogenic fungus that causes serious infections of the central nervous system in individuals immunocompromised by AIDS or undergoing organ transplantation, cytotoxic chemotherapy, or corticosteroid therapy (6, 31). The virulence of C. neoformans is influenced by several factors, including the production of an antiphagocytic polysaccharide capsule (7,20,27), the use of melanin as an antioxidant (26, 51), growth at host physiological temperature (37 to 39°C) (27, 29), and prototrophy (41). Although not directly involved in the virulence of C. neoformans, mating and filamentous growth may play a survival role in the environment and also promote dissemination of the pathogen into the host. Signaling cascades regulating virulence and differentiation of C. neoformans have been extensively studied, including a mitogen-activated protein kinase (MAPK) pathway, a G-protein-regulated cyclic AMP (cAMP) pathway, a Ras-specific pathway, and the calcineurin pathway (for reviews, see references 29 and 48).The MAPK cascade regulates mating processes involving morphological differentiation, such as the dikaryotic mycelia, basidia, and basidiospores, which are produced in response to peptide pheromones secreted by opposite mating-type cells (24,32,43). The MAPK pathway is composed of mating-typespecific (Ste3␣/a, Ste20␣/a, Ste11␣/a, and Ste12␣/a) and nonspecific (Gpb1, Ste7, and Cpk1) elements (12, 49). Gene disruption experiments revealed that the MAPK pathway is required for mating and cell type-specific differentiation but not for virulence (12). However, mating type has been associated with the virulence of serotype D (variety neoformans) strains by Kwon-Chung et al., who showed that the ␣-mating type is more virulent than the a-mating type (25). Furthermore, Del Poeta et al. demonstrated that the MF␣1 pheromone gene is induced during the late stages of central nervous system infection...

Two Major Inositol Transporters and Their Role in Cryptococcal Virulence

“…Niger seed medium was used to test for melanin production. Dulbecco's modified Eagle's (DME) medium for assessing capsule production was prepared as previously described (3). All other media were prepared as described previously (1,64).…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…When Gpa1 is released from the trimeric G protein complex, it activates the adenylyl cyclase Cac1, a protein also reg- ulated by Aca1. Aca1 physically interacts with Cac1 in order to regulate the induction of cAMP (27). Through cAMP production, Cac1 activates the PKA catalytic subunits Pka1 and Pka2.…”

Mating Pheromone in Cryptococcus neoformans Is Regulated by a Transcriptional/Degradative “Futile” Cycle

“…To assess for this, time course mating fusion experiments were conducted in which a H99 ϩ empty vector ϩ NAT (MAT␣) or an H99 ϩ VAD1 ϩ NAT (MAT␣) VAD1 overexpressor strain was crossed with a KN99 ϩ NEO (MATa) strain and colonies were observed on double antibiotic-containing medium as described previously (30). Interestingly, overexpression of VAD1 did not prevent mating (Fig.…”