G-Protein Signaling Mediates Asexual Development at 25°C but Has No Effect on Yeast-Like Growth at 37°C in the Dimorphic Fungus Penicillium marneffei

Abstract

The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25°C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37°C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a G␣ subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a ⌬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°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.Environmental signals leading to a variety of developmental processes and physiological responses are sensed and transduced to the cell interior through complex interactions at the cell membrane. Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal transducers that couple cell surface receptors to cytoplasmic effector proteins in eukaryotes. Upon binding of the agonist with the corresponding serpentine transmembrane receptor, the G␣ subunit becomes activated, exchanging GDP for GTP and dissociating from the ␤␥ complex. G␣ and G␤␥ subunits can then interact with appropriate targets such as phosphodiesterases, protein kinases, adenylyl cyclases, phospholipases, and ion channels to trigger downstream signaling pathways (20,27,37,44).In fungi, G proteins affect a number of developmental and morphogenic processes and play essential roles during pathogenic and sexual development programs (4). The best-characterized fungal G␣ proteins are Saccharomyces cerevisiae Gpa1 and Gpa2. Gpa1 is linked to cell-type-specific pheromone receptors encoded by the STE2 and STE3 genes and plays a negative role in the mitogen-activated protein kinase pheromone signaling pathway that leads to mating (32). Gpa2 is required for the induction of pseudohyphal growth in diploid yeast cells under nitrogen starvation and signals through a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway (30, 35). Interestingly, Gpa2 is coupled to a G-proteincoupled receptor, Gpr1, which appears to be a glucose sensor (28,33,36). G␣ subunits from other fungi with high homology to Gpa2 have also been implicated in morphogenesis through cAMP-PKA signaling. The corn smut fungus Ustilago maydis displays a dimorphic switch between a haploid yeast-like form and a pathogenic filamen...

The GanB Gα-Protein Negatively Regulates Asexual Sporulation and Plays a Positive Role in Conidial Germination in Aspergillus nidulansSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AF198116.

“…Group III fungal G␣-proteins play crucial roles in growth and development, including sporulation When ligands interact with their seven cognate transmembrane receptors, the GDP bound to the ␣-subunit and germination, and pathogenesis in diverse fungi (Liu and Dean 1997;Kays et al 2000;Liebmann et al is replaced with GTP, resulting in dissociation of the ␣GTP-subunit from the ␤␥-complex. The ␣-subunit with 2003; Zuber et al 2003). The cAMP/PKA system is also involved downstream of group III G␣-protein signaling GTP bound and the free ␤␥-complex may interact with many types of downstream effectors such as enzymes in some processes such as cell proliferation, development, stress response, mating, and virulence (Kü bler (adenylate cyclases, phosphodiesterases, phospholipases, etc.)…”

“…Deletion of cyaA, the adenylate cyclase Southern analysis and colony hybridization showed that the gene, resulted in severe delays in germ tube formation, chromosome VIII-specific genomic library (Brody et al 1991) and inactivation of pkaA resulted in slower germ-tube- (Zuber et al 2003).…”

“…The mutants and sequencing the flanking region of inserted amino acid sequence of GanB shows 75-90% identity argB showed that the deletion was successful (data not to the amino acids of GpaB (Liebmann et al 2003) and shown). GasC (Zuber et al 2003) of the human pathogenic fungi…”

“…Transcript levels for ganB were When fungal G␣-proteins are classified according to much higher in these mutants than in the parental amino acid sequence, these G␣-proteins belong to the strains VER7 and RMS011 (data not shown). group closely related to the mammalian G␣ s -subfamily Gln208 in the switch II region and Arg182 in the that is involved in activation of adenylate cyclase (Bölker switch I region of the GTPase domain of G␣ protein 1998; Zuber et al 2002).…”

“…The Gly207Arg mutation prevents conforma-GTP-bound state (Simon et al 1991;Coleman et al tional changes to the GTP-bound G␣-subunit, resulting 1994). Many constitutively active mutants with substituin inhibition of the release of G␤␥; the G protein thus tions of these amino acid residues have been isolated stays in a constitutively inactive state (Kurjan 1992; Yu in a variety of fungal G␣ proteins (Wieser et al 1997;Zuber et al 2003). The difference between Yang and Borkovich 1999; deletion and inactivating mutants of the G␣-protein is 2003).…”