A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

Abstract

The G protein ␣ subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional G␤ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward G␣. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1 Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three G␣ proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1 Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2 Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein ␣ subunits function with or without a canonical G␤ partner in C. neoformans.

The cAMP/protein kinase A signaling pathway in pathogenic basidiomycete fungi: Connections with iron homeostasis

“…1). Ric8 also interacts with the Gα protein Gpa2 that has a role in pheromone signaling and this is consistent with the ric8 Δ mutant phenotype of reduced mating (Gong et al ., 2014). In C. neoformans , pheromone exchange between mating partners activates the Gα protein Gpa2 by dissociating it from a Gβγ complex.…”

“…(2014) reported that the Ric8 protein (resistance to inhibitors of cholinesterase 8) is also involved in the cAMP/PKA pathway and pheromone signaling during mating in C. neoformans (Gong et al ., 2014). Ric8 functions as a guanine nucleotide exchange factor (GEF) for activation of Gα (Gpa1) in the cAMP/PKA pathway and deletion of RIC8 results in capsule and melanin defects similar to those of the gpa1 Δ mutant.…”

“…1). Ric8 interacts with both Gpa1 and Gpa2 and establishes a connection between cAMP signaling via Gpa1 and pheromone signaling via Gpa1 and a MAP kinase pathway (Gong et al ., 2014). A connection between cAMP signaling and another MAP kinase pathway for cell wall integrity has also been established recently in C. neoformans (Donlin et al ., 2014) (Fig.…”

TheCryptococcus neoformansTitan cell is an inducible and regulated morphotype underlying pathogenesis

“…The avirulence of mutants deficient in cAMP signal transduction has precluded direct testing of this model [20,28,29]. We therefore examined the influence of GPA1, CAC1, and PKA1, as well as RIC8, a Gα Guanine Nucleotide Exchange Factor (GEF) for Gpa1, on in vitro Titanisation [30]. Strains deficient in each of these genes failed to generate large cells in our assay ( Fig.…”

“…The gpr4Δ gpr5Δ [42] was kindly provided by Joseph Heitman, Duke University, NC, USA. Strains ric8Δ and usv101Δ were obtained from the Madhani 2015 collection (NIH funding, R01AI100272) from the Fungal Genetics Stock Centre and were validated by PCR and shown to phenotypically match published strains [30,31]. C. gattii R265 [43] was provided by Neil Gow, University of Aberdeen, UK.…”