Canonical Heterotrimeric G Proteins Regulating Mating and Virulence ofCryptococcus neoformans

Abstract

Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three G␣, one G␤, and two G␥ subunit proteins. We have found that G␣s Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3␣, G␤ subunit Gpb1, and RGS protein Crg1. Crg1 also exhibited in vitro GAP activity toward Gpa2. These findings suggest that Gpa2 regulates mating through a conserved signaling mechanism. Moreover, we found that G␥s Gpg1 and Gpg2 both regulate pheromone responses and mating. gpg1 mutants were attenuated in mating, and gpg2 mutants were sterile. Finally, although gpa2, gpa3, gpg1, gpg2, and gpg1 gpg2 mutants were fully virulent, gpa2 gpa3 mutants were attenuated for virulence in a murine model. Our study reveals a conserved but distinct signaling mechanism by two G␣, one G␤, and two G␥ proteins for pheromone responses, mating, and virulence in Cryptococcus neoformans, and it also reiterates that the link between mating and virulence is not due to mating per se but rather to certain mating-pathway components that encode additional functions promoting virulence. INTRODUCTIONCryptococcus neoformans is an encapsulated yeast-like fungus capable of infecting both immunocompromised and healthy individuals to cause life-threatening meningoencephalitis (Mitchell and Perfect, 1995;Chayakulkeeree and Perfect, 2006). The organism belongs to basidiomycota taxonomically and has a defined life cycle and a bipolar mating system with MAT␣ being the predominant mating type in both environmental and clinical settings (Hull and Heitman, 2002;Wang and Fox, 2005;Nielsen and Heitman, 2007). Like other fungi and higher eukaryotes, heterotrimeric G protein-mediated signaling pathways are central for C. neoformans to sense environmental-and host-imposed cues and to respond through regulation of developmental processes such as mating and haploid differentiation, as well as the production of several virulence factors including melanin and capsule (Alspaugh et al., 1998;Wang and Fox, 2005).The lower eukaryotic organisms such as fungi also encode multiple G␣ subunits, similar to higher eukaryotic organisms such as animals and plants . For example, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe encode two G␣ subunits, the plant pathogen Ustilago maydis encodes four G␣ subunits, and many other fungal species contain three G␣ subunits. The S. cerevisiae G␣ Gpa1 is a negative regulator of the pheromone-responsive mating pathway (Whiteway et al., 1989;Whiteway et al., 1990;Clark et al., 1993;Kü bler et al., 1997;Colombo et al., 1998;Thevelein and Winde, 1999), whereas G␣ Gpa2 positively controls a glucose-sensing cAMP pathway for pseudohyphal differentiation and invasive growth (Kü bler et al., 1997;Lorenz and Heitman, 1997). Distinct, but largely similar, signaling pathways mediated by two G␣ subunits were also found in S. pombe (Sti...

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Gpa2 and Gpa3 exhibit shared and distinct functions to collectively regulate pheromone responses, mating, and virulence (237,325). Gpa2 normally couples to the G␤ protein Gbp1 and the G␥ subunit Gpg1 or Gpg2 as a heterotrimer G protein complex.…”

“…Gpa2 normally couples to the G␤ protein Gbp1 and the G␥ subunit Gpg1 or Gpg2 as a heterotrimer G protein complex. The mechanism by which Gpa3 regulates pheromone production and conjugation tube formation remains unclear (325). Gbp1 mediates the response to pheromones in an manner analogous to that of the Ste4-Ste18 dimer in S. cerevisiae.…”

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

“…The cyclic AMP (cAMP)-dependent signaling pathway consists of the G␣ protein Gpa1, the adenylyl cyclase Cac1, the protein kinase A catalytic subunit Pka1 and regulatory subunit Pkr1, the adenylyl cyclaseassociated protein AcaI, and the phosphodiesterases Pde1 and Pde2 (9)(10)(11)(12). The second pathway consists of the G␤ protein Gpb1, which couples with either Gpg1 or Gpg2 as a heterodimer to complex with the G␣ protein Gpa2 as the typical heterotrimeric complex to govern the pheromone-responsive mating pathway (13,14). In C. neoformans, cAMP signaling is also closely linked to the production of virulence factors, such as the melanin pigment and the capsule.…”

Heterotrimeric G-Protein Subunit Function in Candida albicans : both the α and β Subunits of the Pheromone Response G Protein Are Required for Mating

“…Among the G␣ subunits, Gpa1 is implicated in cAMP signaling (1), while Gpa2p and Gpa3p are involved in the response to pheromones. However, this involvement is multifaceted, as the loss of either subunit does not block mating but the loss of both subunits creates a bilateral mating defect and leads to the constitutive expression of mating-factor-induced genes (26,36). Analysis of putative hyperactive alleles suggests that Gpa2p functions positively in response to pheromones and that Gpa3p acts negatively (26); this assessment is supported by the observation that the removal of Gpa3p allows the constitutive activation of the pheromone response genes.…”

“…For example, in Cryptococcus neoformans, there are three genes encoding G␣ subunits, with a single gene for G␤ and two for G␥. The loss of the unique G␤ subunit Gpb1p (60) or one of the G␥ subunits (26,36) blocks mating, suggesting that the ␤␥ subunit acts as a positive regulator of the process. Among the G␣ subunits, Gpa1 is implicated in cAMP signaling (1), while Gpa2p and Gpa3p are involved in the response to pheromones.…”