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. 2007 Nov;18(11):4201–4209. doi: 10.1091/mbc.E07-02-0136

Canonical Heterotrimeric G Proteins Regulating Mating and Virulence of Cryptococcus neoformans

Lie Li *,†,‡, Gui Shen *,†, Zheng-Guang Zhang †,§, Yan-Li Wang †, Jill K Thompson †, Ping Wang *,†,‖,✉
Editor: Daniel Lew
PMCID: PMC2043552  PMID: 17699592

Abstract

Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three Gα, one Gβ, and two Gγ subunit proteins. We have found that Gαs Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3α, Gβ 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 Gγs 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 Gα, one Gβ, and two Gγ 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.

INTRODUCTION

Cryptococcus 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 MATα 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 Gα subunits, similar to higher eukaryotic organisms such as animals and plants (Lengeler et al., 2000). For example, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe encode two Gα subunits, the plant pathogen Ustilago maydis encodes four Gα subunits, and many other fungal species contain three Gα subunits. The S. cerevisiae Gα Gpa1 is a negative regulator of the pheromone-responsive mating pathway (Whiteway et al., 1989; Whiteway et al., 1990; Clark et al., 1993; Kübler et al., 1997; Colombo et al., 1998; Thevelein and Winde, 1999), whereas Gα Gpa2 positively controls a glucose-sensing cAMP pathway for pseudohyphal differentiation and invasive growth (Kübler et al., 1997; Lorenz and Heitman, 1997). Distinct, but largely similar, signaling pathways mediated by two Gα subunits were also found in S. pombe (Stiefel et al., 2004; Hoffman, 2005). Unlike the two yeast models, only the Gα subunit that functions in the conserved cAMP-dependent signaling pathway was better understood in fungi that contain three or more Gαs (Gao and Nuss, 1996; Choi and Dean, 1997; Liu and Dean, 1997; Regenfelder et al., 1997; Ivey et al., 2002; Kays and Borkovich, 2004).

Despite encoding multiple Gαs, only a single Gβ was found in fungi. Extensive genetic and biochemical studies have shown that S. cerevisiae Gβ Ste4 couples with Gγ Ste18 as a protein heterodimer to regulate mating in response to pheromone stimulation. In comparison, the S. pombe Gβ Git5 and Gγ Git11 heterodimer functions in a cAMP-signaling pathway mediated by Gpa2, instead of the mating pathway mediated by Gpa1 (Stiefel et al., 2004). In Neurospora crassa, Gβ Gnb1 and Gγ Gng1 were found to maintain the stability of all three Gαs during multiple signaling, although the molecular basis for such signaling mechanisms is not clear (Yang et al., 2002; Krystofova and Borkovich, 2005).

Previous studies have demonstrated that there exist two distinct G protein–mediated signaling pathways in C. neoformans, similar to those described for S. cerevisiae and S. pombe (Alspaugh et al., 1998; Wang and Heitman, 1999). Gpa1 mediates a conserved cAMP-dependent signaling pathway to regulate morphogenesis and virulence traits (Alspaugh et al., 1997, 2002; D'Souza et al., 2001; Bahn et al., 2004; Xue et al., 2006), and Gpb1 plays a positive role in governing the mating response, such as conjugation tube formation, and mating (Wang et al., 2000). The Gγ subunits Gpg1 and Gpg2 were found to associate with Gpb1 and the Gβ-like/RACK1 (receptor for activated C kinase 1) Gib2, but their functions remain uncharacterized (Palmer et al., 2006).

With two functionally distinct G protein-signaling pathways (cAMP signaling and mating) and a single Gβ subunit, why does C. neoformans encode three Gα and two Gγ subunits? Here we show that Gpa2, Gpa3, Gpg1, and Gpg2 all have a role in pheromone response and mating. We provide evidence suggesting that Gpa2, Gpb1, and Gpg1 or Gpg2 could form a heterotrimeric G protein complex and that Gpa2 regulates mating through a conserved mechanism. Despite interactions detected between Gpa3 and Ste3α, and Gpa3 and Crg1, a functional mechanism for Gpa3 remains to be delineated. Finally, we provide direct evidence demonstrating that Gpa2 and Gpa3, but not Gpg1 and Gpg2, collectively regulate virulence of C. neoformans in a murine virulence model.

MATERIALS AND METHODS

Strains, Media, and Plasmids

C. neoformans consists of two highly related, but distinct varieties (serotypes): var. grubii (serotype A) and var. neoformans (serotype D), with var. grubii being the predominant type. The wild-type MATα (hereafter expressed as α) H99 and MATa (hereafter a) KN99a, as well as their ura- derivatives F99 and F99a, were described previously (Perfect et al., 1993; Nielsen et al., 2003; Wang et al., 2004b). The a ura5 ade2 strain was created by transformation of an F99a strain with the ade2 knockout allele linked to a nourseothricin-resistant marker (NAT1; McDade and Cox, 2001). All of the strains in this study were of the var. grubii, and their genotypes and sources are listed in Supplementary Table 1 (Supplementary Data). Oligonucleotide primers for PCR amplification are listed in Supplementary Table 2 (Supplementary Data).

Yeast extract-peptone-dextrose (YPD), synthetic medium (SD), 10% V8 agar (pH 5.0) for mating, Niger seed agar for melanin production and filament agar for conjugation tube formation were prepared following standard protocols or as described previously (Wang et al., 2000, 2004b).

cDNA for GPA1, GPA2, GPA3, GPG1, and GPG2 was synthesized by RT-PCR and inserted into the pGBKT7 plasmid as described previously (Palmer et al., 2006). GPB1 and CRG1 cDNA was inserted into pGADT7 (BD Biosciences, San Jose, CA). Partial cDNA encoding the C-terminal domains of Ste3α (GenBank AAN75177) and Cpr2 (H99 sequence, homologous to GenBank XM_569248) was synthesized with primers PW347 and PW348, and PW434 and PW435, respectively, and inserted in pGADT7.

Nucleic acid manipulation procedures were performed according to standard protocols (Sambrook and Russell, 2001).

Identification and Disruption of GPA2, GPA3, GPG1, and GPG2 Genes

GPA2, GPA3, GPG1, and GPG2 genomic DNA was identified from the database at http://cneo.genetics.duke.edu (var. grubii), and the sequences were obtained by PCR amplification using primers PW366 and PW117 (GPA2), PW436 and PW141 (GPA3), PW237 and PW249 (GPG1), and PW239 and PW248 (GPG2), respectively. The gpa2::URA5 gene disruption-specific mutation allele was constructed as follows. First, a 1.4-kb fragment containing the GPA2 coding domain was amplified into two partially overlapping fragments that were each ∼700 base pairs in length. The first fragment was amplified using primers PW114 and PW223, and the second fragment was amplified using PW224 and PW117. A SmaI restriction site was incorporated into primers PW223 and PW224, so that the URA5 gene could be inserted into the site creating the gpa2::URA5 disruption allele. The gpa3::URA5 knockout allele was created similarly with the following primers: PW47, PW141, PW51, and PW52. The gpg1::URA5 gene knockout allele was constructed with primers PW237, PW362, PW363, and PW249. The gpg2::URA5 allele was created using primers PW239, PW 364, PW365, and PW248. Mutant alleles were introduced into F99 and F99a strains by biolistic transformation to obtain gpa2, gpa3, gpg1, and gpg2 strains of both α and a types. The gpa2 crg1 and gpa3 crg1 mutant strains were constructed by transformation of the gpa2 (PWC279) and gpa3 (PWC201) mutant strains with the crg1::NAT1 allele. The gpa2 gpa3 double mutant strains were obtained by crossing α gpa3 (PWC201) to a gpa2 (PWC569) followed by microdissection of the basidiospore progenies as described previously (Sia et al., 2000; Palmer et al., 2006).

The α gpa2 and α gpa3 mutant strains were complemented by reintroducing a 3.0-kb fragment containing the wild-type GPA2 gene and a 2.8-kb fragment containing the GPA3 gene linked to the NAT1 marker. The α and a gpg1 and gpg2 mutant strains were complemented similarly.

The gpg1::NAT1 and gpg2::NAT1 alleles were also created to obtain an additional set of gpg1 and gpg2 mutants in α strains. α gpg1 gpg2 mutant strains were obtained by transformation of a gpg2::URA5 strain, PWC424, with the gpg1::NAT1 allele and screened for gpg1::NAT1 gpg2::URA5 transformants. All transformant and segregant strains were verified by PCR amplification and Southern blot hybridization analysis.

Phenotypic Characterization

Pheromone responses were monitored through formation of conjugation tubes that were induced by streaking cells adjacent to those of the opposite mating type on standard filament agar containing 0.5% glucose (Wang et al., 2000). Mating was performed by mixing and patching α and a cells on standard V8 agar. For quantitative assessment of mating, 5 μl of α cells (106 cell/ml) was spotted in triplicate mixing with an equal number of a cells (a ura5 ade2) on synthetic low ammonium dextrose agar (SLAD). After incubation at 22°C for 7 d, colonies were extracted and suspended in sterile water. Cells were then serially diluted and spread on SD-Ade medium containing 5-FOA (5-fluoroorotic acid), and surviving meiotic progenies (ADE2 ura5) were counted as a quantitative measurement for mating efficiency.

Production of melanin was observed by growing cells on standard Niger seed agar at 30°C for 48 h (Wang et al., 2004a,b). The wild-type strains exhibited dark brown pigments, whereas white opaque colonies indicated a defect in melanin production. The polysaccharide capsule was induced by incubating cells in Dulbecco's modified Eagle's medium (DMEM) at 30°C for 48 h, negatively stained with India ink, and visualized using an Olympus microscope (BX51; Melville, NY) equipped with a digital camera (Wang et al., 2004a).

Virulence tests were carried out in female A/JCR and male severe combined immunodeficiency disease (SCID) mice using a murine inhalation model, and mouse survival was analyzed by the Kaplan-Meier method using Prism 4.0 software (GraphPad Software, San Diego, CA), as described previously (Wang et al., 2004b). Animal testing was carried out in compliance with the Institutional Animal Care and Use Committee (IACUC).

Yeast Two-Hybrid, Protein Expression, and Binding Assays

The two-hybrid assay was carried out in a PJ69-4a yeast host as described previously (James et al., 1996; Palmer et al., 2006).

To express Gpa2, Gpa3, and Gpb1 proteins in yeast, cDNA was inserted into the expression vector pYES2/NT, and the corresponding plasmids were transformed into a yeast strain (INVSc1, Invitrogen, Carlsbad, CA). Yeast transformants were grown in liquid SD-Ura medium containing 2% glucose for 24 h at 30°C, and cells were precipitated, washed, and grown in SD-Ura medium containing 2% galactose and 1% raffinose overnight in a 30°C shaker (225 rpm). To extract proteins, cells were collected, suspended in lysis buffer (phosphate-buffered saline [PBS], pH 7.4, 1% Nonidet P-40, and a cocktail of protease inhibitors [Roche, Indianapolis, IN]), mixed with an equal volume of acid-washed glass beads (∼400 μM, Sigma, St. Louis, MO), and homogenized in a Fast-Prep cell disrupter (Bio101, Carlsbad, CA) four times for 40 s at 4°C. Homogenized samples were centrifuged at 13,000 rpm for 30 min at 4°C, and supernatants were recovered. For protein purification, cell extracts were incubated with Co2+ affinity resin (Clontech, Palo Alto, CA) for 1.5 h at 4°C and washed, and proteins were eluted with buffer containing 200 mM imidazole. Proteins were then concentrated through a centrifugal filter (Millipore, Bedford, MA), measured using a BCA protein assay kit (Pierce, Rockford, IL), and verified by SDS-PAGE analysis and Western blotting using the anti-Xpress mAb (Invitrogen).

For protein production in Escherichia coli, cDNA for CRG1, RGS1 (partial CRG1 sequence encoding the RGS domain), GPB1, GPG1, and GPG2 was inserted into the pET41b vector, and plasmid DNA was transformed into Rosetta 2(DE3) cells (Novagen, Madison, WI). Protein expression was induced for 4 h at room temperature after the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. Cells were collected by centrifugation, washed, and stored at −70°C. To extract proteins, cells were suspended in PBS buffer (pH 7.3) also containing 1 mM EDTA, 0.5% Nonidet P-40, 0.5 mg/ml lysozyme, and protease inhibitors (Roche) and incubated for 30 min at 4°C with gentle shaking. Samples were centrifuged at 13,000 rpm for 20 min at 4°C and supernatants were recovered. For protein purification, supernatants were mixed with glutathione-Sepharose resin (Amersham Pharmacia, Piscataway, NJ) for 1.5 h, washed, and proteins eluted with 15 mM glutathione. Proteins were also verified by SDS-PAGE analysis and Western blotting with the anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz, CASC-138). The GST-Gpg1 and GST-Gpg2 fusion proteins were produced using a similar approach.

For binding, 400 μl of the GST-Gpb1 protein extract was added to glutathione-Sepharose resin and washed with buffer C (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM PMSF, and proteinase inhibitors). Gpa2 and Gpa3 proteins were first preconditioned in buffer A (50 mM NaHepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 25 mM β-glycophosphate, 100 μM Na3VO4, 10 μM GDP, and the Roche protease inhibitor cocktail) for 30 min at room temperature before adding to resin containing Gpb1. The protein mixture was incubated overnight with gentle rotation at 4°C, precipitated, and washed twice with PBS buffer containing 0.2% Nonidet P-40 and 1 mM PMSF. SDS-containing gel loading buffer, 50 μl, was added to the resin, and, after a brief boiling, 20 μl was analyzed by SDS-PAGE electrophoresis and Western blotting using the anti-Xpress and -GST antibodies. Buffer B (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10 μM GDP, 30 μM AlCl3, 10 mM NaF, and proteinase inhibitors) was used to promote the transition state of Gαs in the assay for binding between Gα proteins and GST-Rgs1. Binding assays for His6-Gpb1 and GST-Gγ proteins were performed similarly.

GAP Activity Assay

To measure the GAP activity, the steady-state GTP hydrolysis assay was performed following the method described previously (Apanovitch et al., 1998; Versele et al., 1999; Lan et al., 2000). His6-Gpa2 and GST-Crg1 proteins were prepared as described above. Gpa2 (200 nM) and [γ-32P]GTP (7500 cpm/pmol, 2 μM final concentration) were mixed at room temperature in 2× GTP buffer (50 mM Na-HEPES, pH 8.0, 1 mM dithiothreitol [DTT], 1 mM EDTA, 2 μM GTP-γ-S, and 10 mM MgCl2) for 2 min. Crg1 was then added, and samples were withdrawn at 2, 4, 6, and 8 min. Samples (in triplicate) were mixed with 5% charcoal (Fisher Scientific, Pittsburgh, PA) in 50 mM NaH2PO4, pH 2, on ice and precipitated for 15 min at 13,000 rpm. Supernatant (400 μl) was transferred to a scintillation vial, and free phosphates were measured using a liquid scintillation counter (Cobra II, Packard Instrument, Meriden, CT). The mean value was plotted with error bars representing the SEM. Statistical significance was calculated using Student's t test through Prism 4.0 software (GraphPad Software). Assays were repeated twice that yielded comparable results.

RESULTS

Gpa2 and Gpa3 Exhibit Distinct Roles in Pheromone Responses

C. neoformans is divided into two distinct, but highly related, varieties: var. grubii (serotype A) and var. neoformans (serotype D). C. neoformans respond to the presence of the opposite mating partner by producing conjugation tubes that lead to cell fusion and formation of dikaryotic filaments under conditions such as nitrogen deprivation and desiccation (Wang et al., 2000). In var. grubii, conjugation tubes are induced in α strains that carry a mutation in the CRG1 gene (crg1) (Figure 1, bottom row), in contrast to var. neoformans, whose conjugation tubes are readily inducible in the wild-type α strain. No changes were found in α gpa2, α gpa3, or α gpa2 gpa3 mutants when facing a crg1 cells, similar to the wild-type strain (Figure 1, top row). However, conjugation tube formation was seen in the a cells facing α gpa3 or α gpa2 gpa3 mutants, suggesting an increased pheromone production by the α mutants. The large number of conjugation tubes stimulated by the α gpa2 gpa3 mutant suggested that the level of pheromones it produced is equal to that of the α crg1 mutant (Figure 1, top and bottom rows). Inclusion of the GPA3 mutation, but not the GPA2 mutation, in the crg1 mutant blocked conjugation tube formation, indicating that Gpa3 is required for this pheromone response (Figure 1, middle row).

Figure 1.

Figure 1.

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Gpa2 and Gpa3 regulate pheromone responses in C. neoformans. The α and a gpa2 and gpa3 mutant strains were streaked, respectively, on filament agar next to a and α crg1 strains. The α gpa2 crg1 and α gpa3 crg1 mutants were also streaked next to the a crg1 strain. The wild-type α and a strains are H99 and KN99a, respectively. Plates were incubated in the dark for 2 d (48 h) at room temperature, and representative sections were photographed using an Olympus microscope (BX51) equipped with a digital camera. Insets are partial images enlarged by threefold. The shade of conjugation tubes (transparent or dark) resulted from variations in light reflection. The test was repeated at least three times with similar results.

In comparison to α cells, phenotypic changes occurring in a cells as a result of GPA2 and GPA3 mutations were less apparent (Figure 1, middle row). However, our test did reveal that Gpa2 has a positive role in pheromone production, as a reduction of conjugation tube formation was seen in the α crg1 cells facing the a gpa2 mutant, in contrast to the same cells facing the a gpa3, a gpa2 gpa3, and wild-type a strains (Figure 1, middle and bottom rows).

On the basis of these observations, we concluded that Gpa2 and Gpa3 play distinct roles in regulating pheromone response in C. neoformans. Gpa2 plays a positive role and Gpa3 a negative one in pheromone production, and Gpa3 is also required for conjugation tube formation.

Gpa2 and Gpa3 Share a Conserved Regulatory Role and Are Both Required for Normal Mating

Mating in C. neoformans is traditionally assessed by coculturing α and a cells on solid V8 agar and observing formation of mating-specific dikaryotic filaments, basidia, and basidiospores. The α and a gpa2 and gpa3 mutant strains each exhibited a moderate increase in mating when compared with wild-type strains with no visible variation in either the structure or the amount of dikaryotic filaments between the cross (Figure 2, top and bottom rows). To confirm that the cross involving gpa2 and gpa3 strains resulted in increased mating efficiency, a quantitative mating assay was carried out by crossing the mutant strains to an a ade2 ura- (FOAr) strain and detecting Ade2 FOAR progenies as a representation of mating efficiency. After coculture on SLAD medium for 7 d, crosses involving gpa2 and gpa3 strains, respectively, produced 112 and 107 Ade2 FOAR progenies, whereas crosses involving the wild-type α and gpa1, a control strain, generated 38 and 5 Ade2 FOAR strains, respectively.

Figure 2.

Figure 2.

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Gpa2 and Gpa3 regulate mating in C. neoformans. The α gpa2, α gpa3, α gpa2 gpa3, α gpa2 crg1, and α gpa3 crg1 mutants were crossed with the wild-type a strain, and a gpa2, a gpa3, and a gpa2 gpa3 strains were crossed with the wild-type α strain. The α gpa2 crg1 and α gpa3 crg1 strains were also crossed with the wild-type a strain. Plates were also incubated in the dark for 7 d at room temperature, and representative data are shown. Dikaryotic filaments, basidia, and basidiospores were visible in all crosses at day 7, with the exception that no filaments or spores were seen in crosses involving gpb1 or between two compatible gpa2 gpa3 mutants. The test was repeated three times yielding similar results.

Intriguingly, both the α and a gpa2 gpa3 strains exhibited attenuated mating when crossed to a wild-type mating partner, and they were unable to produce any dikaryotic filaments when crossed with each other (Figure 2, top and bottom rows), suggesting Gpa2 and Gpa3 are collectively required for normal mating.

Association between Gα Subunits and Gα-interacting Proteins

To help reveal the mechanism by which Gpa2 and Gpa3 regulate pheromone responses and mating, we utilized the yeast two-hybrid and protein pulldown assays to examine whether Gpa2 and Gpa3 physically interact with proteins known to also play a regulatory role in mating, such as the pheromone receptor homolog Ste3α, Gpb1, and the RGS protein Crg1.

Ste3α/a (Cprα/a) and Cpr2 are the two pheromone receptor homologues in C. neoformans. Mutation of the STE3α/a genes attenuated mating, whereas Cpr2 remains uncharacterized (Chung et al., 2002, 2003; Loftus et al., 2005). We found that Gpa2 interacted with the C-terminal domain of Ste3α (Ste3α-C) in a yeast two-hybrid assay, suggesting that Ste3α could activate Gpa2 in response to pheromone stimulation (Figure 3A). We also found that Gpa3 interacted with Ste3α (Figure 3A), suggesting a similar function, although the interaction was less robust in comparison to that between Ste3α and Gpa2.

Figure 3.

Figure 3.

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Interactions between Gα subunits and Gα-interacting proteins. (A) Gpa2 interacts with Ste3α, Gpb1, and Crg1, whereas Gpa3 only interacts with Ste3α and Crg1 in a yeast two-hybrid assay. GPA1, GPA2, and GPA3 cDNA was inserted into pGBKT7 (BD; Palmer et al., 2006) and cDNA for the C-terminus of Ste3α (Ste3α-C), GPB1, and CRG1 were inserted into pGADT7 (AD). The AD and BD plasmids were cotransformed into yeast PJ69-4a, and transformants were plated on SD-Leu-Trp for 3 d and on selective SD-Leu-Trp-His-Ade, with or without 3-AT, for 5 d. (B) Gpa2, but not Gpa3, interacts with Gpb1 in vitro. His6-Gpa2, -Gpa3, and GST-Gpb1 were expressed and purified by affinity chromatography, and binding was carried out (see text). Bound proteins were separated by SDS-PAGE in duplicate and analyzed by Western blotting. Membrane blots were incubated with the anti-Xpress antibody (top panel) and the anti-GST mAb (bottom panel). The latter blot was scanned instead of autoradiographed for better visibility. Gpa2 and Gpa3 share approximately the same molecular mass of 40 kDa. Lanes 1 and 5 are direct input controls for His6-Gpa2 and His6-Gpa3 (20 μl), respectively.

A previous study has shown that Gpb1 is a positive regulator of pheromone responses and mating in C. neoformans, analogous to Ste4 of S. cerevisiae (Wang et al., 2002). Accordingly, Gpa2 was found to interact with Gpb1 in both in vivo and in vitro assays. As shown in Figure 3A, the growth of the yeast host was robust in the presence of 1 mM 3-amino-1,2,4-triazole (3-AT), suggesting a tight binding. This binding was further demonstrated by an in vitro binding assay in which His6-Gpa2 bound with the GST-Gpb1 fusion protein (Figure 3B, top panel, lane 4), in contrast to that involving the His6-Gpa2 and GST proteins (Figure 3B, lane 3). Surprisingly, an interaction was not found for His6-Gpa3 and GST-Gpb1 in both in vivo and in vitro assays, suggesting that Gpa3 may not couple to Gpb1 for function.

Crg1 Accelerates the GTPase Activity of Gpa2 In Vitro

We previously characterized the RGS protein homolog Crg1 as a negative regulator for mating, because the crg1 mutant strains were enhanced in pheromone response and mating (Nielsen et al., 2003; Wang et al., 2004b). Consistent with the observation that Gpa2 and Gpa3 are mating-specific, we found that they both associate with Crg1 in a yeast two-hybrid assay (Figure 3A). Although the interaction between Gpa3 and Crg1 was less prominent than that between Gpa2 and Crg1, Gpa3, in its transition state (GDP-AlF4 bound), was found to bind to the RGS domain of Crg1 (Rgs1) in a pulldown assay (Figure 4A, top panel, lanes 4 and 8), similar to Gpa2. Thus, these assays indicate that Crg1 could desensitize or inactivate pheromone signaling through direct interaction with Gpa2 and Gpa3.

Figure 4.

Figure 4.

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Both Gpa2 and Gpa3 interact with Crg1 in vitro and Crg1 also accelerates the GTPase activity of Gpa2. (A) His6-Gpa2 and -Gpa3 were expressed in yeast and purified using Co2+ affinity resin chromatography. The Crg1 RGS domain, Rgs1, was expressed in E. coli and purified by glutathione affinity chromatography. After Western blotting, membranes were incubated with either the anti-Xpress antibody (top panel) or the anti-GST antibody (bottom panel). Lanes 1 and 5 are direct input controls for His6-Gpa2 and His6-Gpa3. (B) The steady-state GTPase assay measures the accumulation of free phosphates (Pi) over an extended time period of 8 min. His6-Gpa2 and GST-Crg1 were expressed and purified by affinity chromatography as described in text. Sampling was carried out at 2, 4, 6, and 8 min in triplicate, and mean values were plotted against time, with SEM as error bars. Student's t test was performed for statistical analysis (see text). Controls contain boiled Gpa2 and Crg1. The assay was repeated multiple times with reproducible results.

To determine whether Crg1 also functions as a GTPase-activating-protein (GAP) by accelerating the hydrolysis of GTP-bound Gα to GDP-bound Gα, we measured the GTPase activity of Gpa2, with and without the presence of Crg1. There are two comparable methods to evaluate the RGS proteins GAP function: a single GTP turnover method detecting the transient release of free phosphates upon addition of the RGS protein to a mixture of preconditioned Gα and [γ32P]GTP, and a steady state method measuring the accumulation of free phosphates over an extended time period. We performed the second method as the extended time allowed easier handling for multiple sampling. The recombinant Crg1 (GST-Crg1) and Gpa2 (His6-Gpa2) proteins were purified by affinity chromatography as described in Materials and Methods. As seen in Figure 4B, the intrinsic GTPase activity of Gpa2 appeared to be minimal, whereas addition of the recombinant Crg1 protein prompted an increase in the GTPase activity of Gpa2 by nearly fivefold (p < 0.0005). The GST-Crg1 exhibited a higher than background, but less than Gpa2, GTPase activity (Figure 4B), and the p value was <0.0002 between GST-Crg1 and GST-Crg1 plus His6-Gpa2, suggesting that the activity by Crg1 alone was minimal and not significant (Figure 4B).

Gpg1 and Gpg2 Exhibit Redundant and Distinct Roles in Mating

C. neoformans encodes two Gγs, in comparison to other fungal species in which a single Gγ subunit has been reported (Lengeler et al., 2000). Gpg1 and Gpg2 were shown to interact with Gpb1 in a yeast two-hybrid assay (Palmer et al., 2006), but the interaction was not confirmed by in vitro protein pulldown experiments. Additionally, functions for both Gγs were unknown.

The GST-Gpg1 and GST-Gpg2 fusion proteins were therefore produced and used for binding with His6-Gpb1 through the protein pulldown assay. As shown in Figure 5A, GST-Gpg1 and GST-Gpg2 fusion proteins were able to bind to His6-Gpb1 in vitro (lanes 2 and 3), whereas no binding was found between GST and His6-Gpb1 (lane 6), suggesting that Gpg1 and Gpg2 can both couple to Gpb1.

Figure 5.

Figure 5.

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Gpg1 and Gpg2 share a role in mating of C. neoformans. (A) Gpg1 and Gpg2 interact with Gpb1 in vitro. His6-Gpb1 was expressed in yeast using the pYES2/NT vector, and Gpg1 and Gpg2 were expressed as the GST-fusion proteins in E. coli. Binding and detection were as the same as those described in Figures 3B and 4A. Lane 1 is the His6-Gpb1 direct input control (20 μl). Lanes 2 and 3 are the same. (B) α gpg1 and α gpg2 mutants were streaked next to a crg1 cells, and a gpg1 and a gpg2 mutant strains to α crg1 cells on filament agar. Plates were incubated in the dark at room temperature for 48 h. The confrontation test between α crg1 and a wild type is from Figure 1. Insets show the images enlarged by 2.5-fold. (C) The α gpg1, α gpg2, and corresponding complemented strains, as well as the α gpg1 gpg2 mutant, were crossed to the wild-type a strain. The a gpg1, a gpg2, and corresponding complemented strains were crossed to the wild-type α strain. Plates were incubated in the dark for 7 d at room temperature. Dikaryotic filaments, basidia, and basidiospores were visible in crosses involving gpg1, but not gpg2 or gpg1 gpg2 mutants.

Moreover, the gpg1 and gpg2 mutants of both α and a mating types, and the α gpg1 gpg2 mutants were created. In confrontation testing, mutation of the GPG1, GPG2, and GPG1 GPG2 genes in α cells did not reveal any detectable phenotypic changes associated with pheromone responses (Figure 5B, top row). However, confrontation of the a gpg1 and a gpg2 mutants to α crg1 showed, respectively, less and short (a gpg1) or barely visible (a gpg2) conjugation tubes in α crg1, indicating that both Gpg1 and Gpg2 play a positive role in pheromone production, with the role for Gpg2 being more prominent (Figure 5B, bottom row).

Consistently, the gpg1 mutant strains showed attenuated formation of mating-specific dikaryotic filaments and basidia, whereas the gpg2 and gpg1 gpg2 (α) mutant strains were sterile when crossed to strains of the opposite mating type (Figure 5C). A small amount of mating-specific structures was still seen when α gpg2 was crossed to a crg1, or a gpg2 crossed to α crg1, after an incubation period of 2 wk or more (data not shown), suggesting that the gpg2 mutant is conditionally sterile. However, no mating structures were seen in the cross involving the gpg1 gpg2 strain, indicating that the gpg1 gpg2 mutant is sterile, similar to that of Gpb1. Results from quantitative mating by crossing the Gγ mutants to the a tester strain (ade2 ura-) yielded 13 (WTα), 9 (gpg1), 2 (gpg2), and 0 (gpg1 gpg2) progenies after a 3-wk incubation on SLAD medium, which were consistent with those from the plate assay.

Individual Mating-specific G Proteins Are Not Required for Virulence

The previous study of C. neoformans Gpb1 demonstrated uncoupling between mating and virulence (Wang et al., 2000). To investigate whether Gpa2, Gpa3, Gpg1, and Gpg2 proteins play any role in virulence, the corresponding mutant and complemented strains were examined for the production of virulence factors: melanin and capsule, and also virulence using a murine model. All gpa2, gpa3, gpa2 gpa3, gpg1, gpg2, and gpg1 gpg2 mutants exhibited normal melanin formation on Niger seed agar (Figure 6A, α strains shown), and none of the mutants showed any abnormal formation of capsule after standard incubation of 48 h in liquid DMEM medium (Figure 6B, also α strains).

Figure 6.

Figure 6.

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Mating-specific G protein subunits do not regulate virulence factors melanin and capsule formation in vitro. (A) The α gpa2, gpa3, gpa2 gpa3 (PWC 573), gpg1 (PWC564), gpg2 (PWC509), and gpg1 gpg2 mutants were spotted on Niger seed agar, and plates were incubated at 30°C for 3 d before being photographed. The gpa1 mutant is defective in melanin production as its colony appeared pigment free. (B) The same set of mutant strains plus gpa1 were grown in liquid DMEM medium for 2 d at 30°C (225 rpm), and cells were stained with India ink. The gpa1 mutant is also defective in capsule formation and serves as a negative control. The capsule appears as a “white” halo surrounding the cell and the image was documented using an Olympus microscope (BX51) and a digital camera. The scale for magnification is indicated.

For direct virulence assessment, the α gpa2, gpa3, gpg1, and gpg2 mutant strains were each introduced into five female A/JCR mice by the nasal route of inoculation, and mouse survival was monitored twice daily after infection. In the first test, mice infected with the wild-type strain had a survival of 23 d, whereas mice infected with gpa2 and gpa3 survived 20 and 22 d, respectively (Figure 7A). A second test revealed that mice infected with gpg1, gpg2, and wild type had survived 27, 25, and 24 d, respectively (Figure 7B). The survival was analyzed using the Kaplan-Meier method and p values were estimated to be 0.5 (gpa2 vs. wild type), 0.5 (gpa3 vs. wild type), 0.25 (gpg1 vs. wild type), and 0.6 (gpg2 vs. wild type), indicating similar survivals (Figure 7B). In the third test that involved male mice with SCID, animals infected with gpa2, gpa3, gpg1, gpg2, gpg1 gpg2, and the wild-type control all had similar survival times (21, 22, 20, 21, 21, and 21, respectively). Mice infected with two independent gpg1 gpg2 knockout mutants (nos. 53 and 55) had an identical survival (Figure 7C, no. 55, not shown). However, mice infected with two independent gpa2 gpa3 mutant segregants (nos. 22 and 23) showed longer survival (25 and 27 d, respectively, Figure 7C), in comparison to the wild-type control (21 d, gpa2 gpa3 vs. WT, p < 0.05, Figure 7C), indicating that Gpa2 and Gpa3 collectively regulate fungal virulence.

Figure 7.

Figure 7.

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Gpa2 and Gpa3 collectively regulate fungal virulence. Mice were inoculated via nasal cavities, and mouse survival was monitored twice daily and plotted against time (Wang et al., 2004b). (A and B) α gpa2, gpa3 (PWC 573), gpg1 (PWC564), gpg2 (PWC509), and wild-type control were each inoculated into five 5-wk-old female A/JCR mice via nasal cavities. (C) α gpa2, gpa3, gpa2 gpa3 (PWC572 and PWC573), gpg1 (PWC564), gpg2 (PWC509), gpg1 gpg2 (PWC574, PWC575 not shown), and wild-type strains were inoculated into five 6-wk-old male SCID mice via nasal cavities. Survival was analyzed using the Kaplan-Meier method (see text).

DISCUSSION

Two Gα Subunits Regulate Pheromone Responses and Mating

In C. neoformans, Gα Gpa1 and Gβ Gpb1 govern a conserved cAMP-dependent signaling and a pheromone responsive mating pathway, respectively (Alspaugh et al., 1998; Wang and Heitman, 1999). Gpa1 did not interact with Gpb1, but it interacted with a Gβ-like/RACK1 protein homolog, Gib2, that regulates cAMP production in strains defective for Gpa1 signaling (Palmer et al., 2006). Although it is apparent that Gpa1- and Gpb1-mediated signaling pathways are analogous to those of Gpa2 and Gpa1 in S. cerevisiae and S. pombe, it is not known whether or which of the remaining two Gαs, Gpa2 or/and Gpa3, functions in mating by coupling to Gpb1. Our study revealed that both Gpa2 and Gpa3 appear to have a role in regulating pheromone responses and mating.

The mechanism by which Gpa2 and Gpa3 regulates the pheromone response and mating is apparently complicated, and this complexity is further compounded by the inherent difference between α and a cells in sensing and responding to pheromones (Figure 1, bottom row). Nevertheless, studies of α cells allowed us to show that Gpa3 has a negative role in pheromone production, and Gpa3 is required for conjugation tube formation. Accordingly, studies of a cells revealed that Gpa2 plays a positive role in pheromone production. The abundance of conjugation tubes in a crg1 stimulated by α gpa2 gpa3, suggesting an enhanced pheromone production and the opposite effect on mating by single and double Gα mutation are all indicative of cross-talk between Gpa2- and Gpa3-mediated pathways. A simplified view for how Gpa2 and Gpa3 might function to regulate pheromone response and mating is given in Figure 8.

Figure 8.

Figure 8.

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Canonical heterotrimeric G protein(s) regulating pheromone responses, mating, and virulence in C. neoformans. The pheromone receptor Ste3α/a is likely to be activated by pheromones that in turn activate the heterotrimeric G proteins Gpa2 and Gpa3. Gpa2 couples to Gpb1 and Gpg1/Gpg2 as a heterotrimer G protein complex, whereas Gpa3 may or may not interact with Gpb1-Gpg1/Gpg2 for mating. Crg1 provides a mechanism for pheromone desensitization and inactivation of mating. The mechanisms by which Gpa2 inhibits, Gpa3 promotes, pheromone production, Gpa3 is required for conjugation tube formation, and Gpa2 and Gpa3 are collectively required for normal mating and virulence remain to be defined.

Gpa2 Regulates Mating through a Conserved Functional Mechanism

Mating assays of both α and a gpa2 and gpa3 mutant strains revealed an increase in mating efficiency, suggesting that Gpa2 and Gpa3 couple to Gpb1 and that mutation of one of the Gα subunits could activate Gpb1 and the conserved downstream MAP kinase pathway. Also, previous studies have shown that other Gα-interacting proteins, such as the pheromone receptor Ste3α and the RGS protein Crg1, also regulate the pheromone responses and mating in C. neoformans (Fraser et al., 2003; Nielsen et al., 2003; Wang et al., 2004b). Protein–protein binding assays were therefore carried out to test possible interactions between Gpa2 and Gpa3 and the above-mentioned proteins. Consistent with a role for Gpa2 in regulating mating through a conserved signaling mechanism, it interacted with Ste3α, Gpb1, and Crg1. Moreover, Crg1 exhibited in vitro GAP activity toward Gpa2.

Intriguingly, despite interacting with Ste3α and Crg1, Gpa3 was not found to interact with Gpb1, suggesting that Gpa3 may function independent of Gpb1, or alternately, the in vivo and in vitro conditions do not foster an environment in which Gpa3 and Gpb1 can interact. We have recently obtained evidence indicating that the GDP-AlF4 bound Gpa3, but not Gpa2, interact with a RGS protein homolog involved in the Gpa1-cAMP pathway of C. neoformans (Shen and Wang, unpublished observations). These observations indicate that Gpa3 may be involved in multiple signaling processes through either conserved or unique functional mechanisms.

Redundant and Unique Functions in Mating by Gpg1 and Gpg2

We previously identified Gpg1 and Gpg2 and showed that both proteins can associate with Gpb1 in a yeast two hybrid assay (Palmer et al., 2006). Here, we show that both Gpg1 and Gpg2 can interact with Gpb1 in vitro and they share a positive regulatory function in pheromone production and mating.

Why does C. neoformans encode two Gγ subunits, whereas other fungi studied so far contain only a single Gγ subunit? One can hypothesize that two Gγ subunits are the product of a gene duplication event and that both Gγs have evolved to assume distinct as well as overlapping functions. A similar proposition was made for the two cyclophilin proteins, Cpa1 and Cpa2, in this fungus (Wang et al., 2001). However, unlike Cpa1 and Cpa2 that are inversely located within a distance of 21 kb on chromosome 2, Gpg1 and Gpg2 are found separately on chromosomes 4 and 7. Regardless, our study has shown that both Gpg1 and Gpg2 couple to Gpb1 and that the Gpb1-Gpg2 heterodimer plays a more prominent role in the activation of the pheromone-responsive mating pathway.

Coregulation between Mating and Virulence

One primary reason to study the mating regulatory mechanism in C. neoformans is that there is a close link between mating type and virulence. Kwon-Chung et al. (1992) reported that a var. neoformans α strain was approximately three times more virulent than a congenic a strain in an experimental murine virulence model. Such an association was further corroborated by studies of mating type locus components, such as Ste20α, Ste11α, and Ste12α/Ste12a, whose mutation attenuated fungal virulence (Chang et al., 2000; Clarke et al., 2001; Wang et al., 2002). Mutation of nonmating type specific components that regulate mating, such as Gpb1 and the MAP kinase homolog Cpk1, resulted in no alteration in virulence (Wang et al., 2000; Davidson et al., 2003). Interestingly, on the other end of the spectrum, mutation of Crg1 resulted in an enhancement in both mating and virulence in an archetypical var. grubii strain (Wang et al., 2004b). However, a similar increase in virulence was not found in the a crg1 strains or the var. neoformans α and a crg1 mutant strains (Thompson and Wang, unpublished observation).

Neither the Gα nor Gγ subunits under study are mating type specific. The in vitro production of melanin and capsule of mutants also did not reveal any significant changes when compared with wild-type strains, suggesting that virulence traits were not affected by gene mutation. In vitro virulence traits often provide a good indication for virulence, but they do not always correspond fully with results from animal models. For example, mutation of the Akt/PKB type protein kinase Sch9 resulted in enlarged capsules in vitro, but the sch9 mutant was severely attenuated in virulence (Wang et al., 2004a). Therefore, the Gα and Gγ mutant strains were subjected to direct virulence testing in murine models. We have shown that individual Gα and individual and collective Gγ subunits do not play any role in virulence. However, Gpa2 and Gpa3 are collectively required for full virulence expression, because two independent gpa2 gpa3 mutants were attenuated in virulence involving male SCID mice (Figure 7C). Thus, the association between mating and virulence appears not due to mating per se but rather to certain components of the mating pathway(s) that encode functions in addition to their conserved role for mating. As a result of mating pathway alteration, decreased/increased activities of mating-associated protein kinases and transcription factors resulted in reduced/enhanced production of the factors that promote virulence.

In summary, C. neoformans is a unique organism in which pheromone responses and mating are regulated by two Gα and two Gγ subunits, as well as a single Gβ subunit. We found that Gpa2 functions through a conserved signaling mechanism and that there may exist an additional regulatory mechanism for Gpa3. Finally, Gpa2 and Gpa3 collectively regulate virulence even though individual Gpa2 and Gpa3 do not. Studies of these G proteins could therefore lead to further revelations of the complexity by which G protein signaling pathways govern the physiology and virulence of this pathogenic fungal organism.

Supplementary Material

[Supplemental Material]
mbc_E07-02-0136_index.html (816B, html)

ACKNOWLEDGMENTS

We thank J. Cutler, D. Fox, G. Olson, and A. Whittington for editorial comments and B. Buggele, S. Martin, and A. Prat for technical assistance. This research was supported in part by a National Institutes of Health Grant AI054958 and funds from the Research Institute for Children, New Orleans, LA.

Abbreviations used:

GAP

GTPase-activating protein

RGS

regulator of G protein signaling.

Note added in proof

We acknowledge online publication of an independent study by Hsueh et al. (2007) on a similar subject before the final acceptance of this study.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-02-0136) on August 15, 2007.

Inline graphic The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

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