G-protein signaling mediates asexual development at 25 degrees C but has no effect on yeast-like growth at 37 degrees C in the dimorphic fungus Penicillium mameffei

Abstract

The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25 degrees C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37 degrees C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a G alpha subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a delta gasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA(G42R)) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA(G203R) mutant shows inappropriate brlA expression and conidiation. Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37 degrees C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.

FIG. 1.

The gasA gene of P. marneffei encodes a Gα subunit of a heterotrimeric G protein. (A) Gene structure and partial restriction map of the gasA gene corresponding to pSZ4809. The region predicted to encode the GasA protein is indicated by gray boxes representing the exons, interrupted by three introns. The solid black arrow shows the direction of transcription. (B) Relationship of fungal Gα subunits. The tree is based on amino acid sequence alignments and was constructed as described in Materials and Methods. Abbreviations of species and GenBank gene accession numbers are as follows: An-FadA, A. nidulans FadA (Q00743); An-GanA, A. nidulans GanA (AF142058); An-GanB, A. nidulans GanB (AF198116); Ca-Cag1, Candida albicans Cag1 (M88113); Ch-Cga1, Cochliobolus heterostrophus CGA1 (AF070446); Cn-Gpa1, Cryptococcus neoformans Gpa1 (AAD46575); Cp-Cpg1, C. parasitica CPG1 (L32176); Cp-Cpg2, C. parasitica CPG2 (L32177); Mg-MagA, M. grisea MagA (AF011340); Mg-MagB, M. grisea MagB (AF011341); Mg-MagC, M. grisea MagC (AF011342); Nc-Gna1, N. crassa GNA1 (L11453); Nc-Gna2, N. crassa GNA2 (L114532); Nc-Gna3, N. crassa GNA3 (AF281862); Pm-GasA, P. marneffei GasA (AF448796); Sc-Gpa1, S. cerevisiae Gpa1 (AAA34650); Sc-Gpa2, S. cerevisiae Gpa2 (AAA34651); Sp-Gpa1, Schizosaccharomyces pombe Gpa1 (M64268); Sp-Gpa2, S. pombe Gpa2 (D13366); Uh-Fil1, Ustilago hordei Fil1 (U76672); Um-Gpa1, U. maydis Gpa1 (U85775); Um-Gpa2, U. maydis Gpa2 (U857756); Um-Gpa3, U. maydis Gpa3 (U85777); Um-Gpa4, U. maydis Gpa4 (U85778n). Hatched lines indicate nonproportional length.

FIG. 2.

Northern blot analysis of gasA expression. Total RNA from the P. marneffei wild-type strain (FRR2161) was isolated from yeast cultures (37°C), vegetative mycelia grown in liquid at 25°C for 2 days (25°C veg), and asexually developing cultures (25°C dev), as indicated in Material and Methods. RNA from each of the three growth stages (37°C, 25°C veg, and 25°C dev) was hybridized with probes specific for either gasA or histone H3 (H3), which was used as a loading control. Arrows indicate the two gasA transcripts of 1.1 and 1.3 kb.

FIG. 3.

Analysis of the gasA^G42R, gasA^G203R, and ΔgasA strains at 25°C. (A) Partial restriction map of the dominant activated gasA^G42R (pSZ5030) and dominant interfering gasA^G203R (pSZ5089) alleles used to generate the respective eponymous strains. The gasA locus in the ΔgasA strain is shown after insertion of the gasA deletion construct (pSZ5031). Hatched lines indicate nonproportional length. (B) Colonial morphologies of the P. marneffei wild-type (FRR2161), dominant activated gasA^G42R, dominant interfering gasA^G203R, and ΔgasA strains. The strains were grown on ANM-GABA for 14 days at 25°C. All strains conidiated normally except for the gasA^G42R mutant, which is aconidial and exhibited flat colony morphology with thickaerial hyphae. (C) Microscopic examination of the P. marneffei wild-type (FRR2161), dominant activated gasA^G42R, dominant interfering gasA^G203R, and ΔgasA strains. The strains were grown on SD containing 0.1% glucose for 3 days at 25°C. All strains exhibited wild-type conidiophores except for the gasA^G42R mutant, which showed no signs of asexual development. Scale bars, 20 μm. Arrowheads highlight the conidiophore structures (M, metulae; P, phialides; C, conidia). (D) Northern blot analysis of the P. marneffei wild-type strain (FRR2161) and the dominant activating gasA^G42R mutant. Total RNA was isolated from vegetative mycelia grown in liquid at 25°C for 2 days (veg) and from asexually developing cultures (dev). Northern blots were probed with either brlA or histone H3 (H3). WT, wild type.

FIG. 4.

Analysis of the gasA^G203R and ΔgasA strains in liquid cultures at 25°C. (A) Microscopic examination of the P. marneffei wild-type (FRR2161), dominant interfering gasA^G203R, and ΔgasA strains. The strains were grown in liquid at 25°C for 3 days. Scale bars, 20 μm. Arrowheads highlight the conidiophore structures (M, metulae; P, phialides; C, conidia). (B) Northern blot analysis of the P. marneffei wild-type (FRR2161), dominant interfering gasA^G203R, and ΔgasA strains. Total RNA was isolated from vegetative mycelia grown in liquid at 25°C for 3 days (veg) and from asexually developing cultures (dev). Northern blots were probed with either brlA or histone H3 (H3). WT, wild type.

FIG. 5.

Analysis of the gasA^G42R, gasA^G203R, and ΔgasA strains at 37°C. (A) Colonial morphologies of the P. marneffei wild-type (FRR2161), dominant activated gasA^G42R, dominant interfering gasA^G203R, and ΔgasA strains. The strains were grown on SD medium at 37°C for 4 days. All strains exhibited normal yeast-like colonies except for the ΔgasA strain, which showed smaller colonies. This phenotype was found not to result from the inactivation of gasA (see the text). (B) Microscopic examination of the P. marneffei wild-type (FRR2161), dominant activated gasA^G42R, dominant interfering gasA^G203R, and ΔgasA strains. The strains were grown on BHI broth at 37°C for 4 days. All strains exhibited wild-type yeast cells. Scale bars, 20 μm. Differential interference contrast (DIC) and DAPI-stained epifluorescence of nuclei (DAPI) are shown. (C) Northern blot analysis of the P. marneffei wild-type (FRR2161), dominant activating gasA^G42R, dominant interfering gasA^G203R, and ΔgasA strains. Total RNA was isolated from yeast cultures grown in liquid at 37°C for 6 days. Northern blots were probed with either brlA, abaA, or histone H3 (H3). WT, wild type.

FIG. 6.

Proposed model for signal transduction pathways regulating development and growth in P. marneffei. GasA signaling at 25°C involves a cAMP-PKA pathway in the regulation of asexual development and the expression of the core regulatory factors BrlA and AbaA. GasA has no apparent role during yeast cell morphogenesis at 37°C. Therefore, we hypothesize the presence of a brlA repressor (black box) as well as an abaA activator (white box) which are independent of GasA signaling at this temperature.