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 Galpha 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.

FIG. 1.

Aca1 associates with adenylyl cyclase and itself. (A) The amino acid sequences of the N-terminal region of adenylyl cyclase-associated proteins (upper) or the C-terminal region of adenylyl cyclases (lower) from S. cerevisiae (ScCap and ScCyr1), S. pombe (SpCap and SpCyr1), C. albicans (CaCap1 and CaCdc35), and C. neoformans (CnAca1 and CnCac1) were aligned. Hydrophobic residues in the heptad motif are in boldface. Asterisks indicate leucine residues essential for interactions between Cap and Cyr1 in S. cerevisiae. (B) Two-hybrid assay showing interactions of Aca1 with Cac1 or itself. Plasmids expressing full-length Aca1, Gpa1, and Ras1, and the C terminus (2126 to 2260 aa) of Cac1 with either the Gal4 DNA-binding domain (BD) or activation domain (AD) were: AD (pGAD424), BD (pGBT9), AD-Aca1 (pGAD-ACA1), BD-Aca1 (pGBT-ACA1), BD-Cac1 (pGBT-CAC1_2126-2260), BD-Gpa1 (pGBT-GPA1), and BD-Ras1 (pGBT-RAS1). Combinations of the indicated plasmids were cotransformed into reporter strain PJ69-4A and three independent transformants isolated from SD−Leu−Trp medium were further grown on SD−Leu−Trp−His or SD−Leu−Trp−His−Ade to test for protein-protein interactions that allow reporter-dependent cell growth and photographed after 48 h of incubation at 30°C.

FIG. 2.

Disruption and reintegration of the ACA1 gene. (A) The strategy for the ACA1 disruption and reintegration showing the genomic DNA structure of ACA1 illustrated as black boxes for the first 8 exons and an arrow for exon 9 indicating the direction of transcription are depicted. The transcription start site (26 bp upstream from the ATG) and terminator regions (83-bp region downstream from the stop [TAG] codon) were determined by 5′ and 3′ RACE (see Materials and Methods). Primers for overlap PCR and diagnostic PCR are indicated as bent arrows. The middle and lower diagrams depict the aca1Δ::NAT disruption allele and the aca1Δ+ACA1 reconstituted allele. (B) Southern blot analysis with PstI-digested genomic DNA of wild-type α and a (lanes 1 and 2), α and a aca1Δ mutants YSB6 and YSB58 (lanes 3 and 4), and α and a aca1Δ+ACA1 reconstituted strains YSB117 and YSB118 (lanes 5 and 6) was performed with the ACA1-specific probe described in panel A. (C) RT-PCR analysis. The absence or presence of the ACA1 message was indicated by a 418-bp RT-PCR product. A 1,223-bp RT-PCR product representing the CAC1 mRNA served as a loading control. Lanes: 1 and 4, wild-type α and a; 2 and 5, α and a aca1Δ mutants YSB6 and YSB58; 3 and 6, α and a aca1Δ+ACA1 reconstituted strains YSB117 and YSB118.

FIG. 3.

Aca1 promotes cell fusion and filament formation during mating but is not required for pheromone production. (A) Serotype A MATα and MATa strains were cocultured on V8 media (pH 5.0) for up to 4 weeks at room temperature in the dark, including H99 and KN99 (α × a), YSB6 and KN99 (aca1 × a), YSB6 and YSB58 (aca1 × aca1), and YSB117 and YSB118 (aca1+ACA1 × aca1+ACA1). Edges of the mating patches were photographed at a magnification of ×100 (inserts in the second row at ×200). (B) Cell fusion assays were performed (see Materials and Methods) with the following strains: YSB119 and YSB121 for α × a, YSB119 and YSB58 for α × aca1, YSB6 × YSB58 for aca1 × aca1, YSB119 × YSB85 for α × gpa1, YSB83 × YSB85 for gpa1 × gpa1, YSB119 × YSB79 for α × cac1, YSB42 × YSB79 for cac1 × cac1, YSB119 × YSB73 for α × ras1, YSB51 × YSB73 for ras1 × ras1, YSB119 × YSB76 for α × gpb1, and YSB49 × YSB76 for gpb1 × gpb1. In each experiment, the percentage of cell fusion relative to the α × a mating (100%) was calculated by averaging results from duplicate plates for three independent experiments with the standard deviations, as indicated. (C) Each diploid strain (WT from YSB119 × YSB121, aca1/aca1 from YSB6 × YSB58, gpa1/gpa1 from YSB83 × YSB85, and cac1/cac1 from YSB42 × YSB79) recovered from the cell fusion assays was grown on YPD medium containing nourseothricin and G418 for 5 days at room temperature and photographed (×100 magnification). (D) MATα crg1Δ mutants were confronted with the MATa crg1Δ mutant as a control or the crg1Δ aca1Δ double-mutant strains on filamentation agar for 1 week at room temperature in the dark and photographed (×100 magnification).

FIG. 4.

Aca1 regulates mating through the Cac1-cAMP-PKA-dependent signaling pathway. (A) Serotype A wild-type strain H99 (α) was cocultured on V8 medium (pH 5.0) with or without 1 or 10 mM cAMP with wild-type strain KN99 (a) or the following MATa mutants for unilateral matings: YSB58 (aca1Δ), YSB85 (gpa1Δ), YSB79 (cac1Δ), YSB73 (ras1Δ), and YSB76 (gpb1Δ). For bilateral matings, MATa mutant strains described above were cocultured in the same conditions with the following MATα mutant strains: YSB6 (aca1), YSB83 (gpa1), YSB42 (cac1), YSB51 (ras1), and YSB49 (gpb1). (B) The serotype A MATα and MATa strains H99 and KN99 (α × a), YSB188 and KN99 (pka1 × a), YSB200 and KN99 (pka1 pka2 × a), YSB188 and YSB191 (pka1 × pka1), and YSB194 and YSB198 (pka2 × pka2) were cocultured on V8 medium (pH 5.0) at room temperature in the dark for 2 weeks and photographed (×100 magnification).

FIG. 5.

Aca1 regulates capsule production via the Cac1-cAMP-signaling pathway. (A) The wild-type H99 (WT), YSB49 (gpb1Δ), YSB51 (ras1Δ), YSB6 (aca1Δ), YSB117 (aca1Δ+ACA1), YSB174 (aca1Δ ras1Δ), YSB83 (gpa1Δ), YSB166 (aca1Δ gpa1Δ), YSB42 (cac1Δ), YSB170 (aca1Δ cac1Δ), YSB188 (pka1Δ), and CAP59 (cap59Δ) strains were grown on agar-based DME media with or without 10 mM cAMP at 37°C for 24 h and resuspended in distilled H₂O. Capsule was visualized by staining with India ink and observed by microscopy. Bar, 10 μm. (B) Quantitative measurements of the relative capsule diameter (see Materials and Methods). A total of 30 to 40 cells (15 to 20 cells from two independent experiments) were measured for each strain, and error bars indicate the standard deviations. The relative capsule size of aca1Δ, gpa1Δ, cac1Δ, or pka1Δ mutant strains was significantly smaller than that of WT, ras1Δ, or gpb1Δ mutant strains (P < 0.001). cac1Δ, aca1Δ cac1Δ, and aca1Δ gpa1Δ mutant strains showed equivalent relative capsule sizes (P > 0.05).

FIG. 6.

Aca1 regulates melanin production in a cAMP-dependent manner but in parallel with Gpa1. (A) The wild-type H99 (WT), YSB6 (aca1Δ), YSB117 (aca1Δ+ACA1), YSB83 (gpa1Δ), YSB42 (cac1Δ), YSB166 (aca1Δ gpa1Δ), YSB170 (aca1Δ cac1Δ), YSB188 (pka1Δ), YSB194 (pka2Δ), YSB200 (pka1Δ pka2Δ), and CHM3 (lac1Δ) (Table 1) strains were grown for 16 h at 30°C in YPD medium and then spotted onto Niger seed medium with or without 10 mM cAMP at 30 or 37°C for 3 days and photographed. (B) For quantitative measurement of laccase activity, 10⁸ cells of the same isogenic strain series in panel A were grown at 30°C for 16 h in l-DOPA medium, transferred to 25°C, and then further incubated for 6 h. The OD₄₇₅ of the culture supernatant was determined. One unit of laccase was defined as an OD₄₇₅ of 0.001. Solid bars present the average from three independent experiments, and error bars show the standard deviations from the mean.

FIG. 7.

Aca1 is required for an increase in cAMP levels in response to glucose readdition. The wild-type (▪, H99), aca1Δ mutant (□, YSB6), aca1Δ+ACA1 reconstituted strain (○, YSB117), gpa1Δ mutant (•, YSB83), aca1Δ gpa1Δ mutant (, YSB166), and cac1Δ mutant (▵, YSB42) were glucose starved for 2 h. After the readdition of glucose, a portion of cell suspension was extracted, and its intracellular cAMP concentration was measured at the indicated times as described in Materials and Methods. Each datum point and error bar indicate the mean and standard deviation, respectively, for duplicate samples of two independent experiments. At all time points except zero, the cAMP levels were significantly higher in the wild-type or aca1Δ+ACA1 reconstituted strains than in the aca1Δ, gpa1Δ, aca1Δ gpa1Δ, or cac1Δ mutants (P < 0.05, as analyzed by using the Bonferroni multiple comparison test).

FIG. 8.

Aca1 is required for virulence of serotype A C. neoformans. A/Jcr mice were infected with 10⁵ cells of MATα wild-type (▪; H99, six mice), aca1Δ (□; YSB6, nine mice), aca1Δ+ACA1 reconstituted (•; YSB117, ten mice), and cac1Δ mutant (; YSB42, ten mice) strains by intranasal inhalation. The percent survival was monitored for 40 days postinfection. The median survival for animals infected with each strain was 23 days for the WT strain and the aca1Δ+ACA1 reconstituted strain and >40 days for the cac1Δ and the aca1Δ mutant strains.

FIG. 9.

Model of the signaling pathways regulating the virulence and differentiation of C. neoformans. Both the MAPK and the cAMP-signaling pathways control mating. The MAPK pathway mediates pheromone-responsive mating signals through the seven transmembrane pheromone receptors Ste3α/a, the Gβ subunit Gpb1, the p20-activated protein kinase Ste20α/a, the MAPK kinase kinase Ste11α/a, the MAPK kinase Ste7, and the MAPK Cpk1. Ras1 appears to transmit mating signals through Gpb1 and downstream MAPK components. In contrast, nutritional starvation signals are transduced via the Gα subunit Gpa1, the adenylyl cyclase Cac1, cAMP, and the catalytic subunits Pka1/2 and regulatory subunit Pkr1 of PKA. Aca1 appears to activate the Cac1-cAMP pathway independent of Gpa1. Solid lines or arrows indicate the major paths of signal flow and dashed arrows represent less significant events.