Dataset Information

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

ABSTRACT: The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

SUBMITTER: Alspaugh JA

PROVIDER: S-EPMC118042 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

Alspaugh J Andrew JA Pukkila-Worley Read R Harashima Toshiaki T Cavallo Lora M LM Funnell Deanna D Cox Gary M GM Perfect John R JR Kronstad James W JW Heitman Joseph J

Eukaryotic cell 20020201 1

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by tra ...[more]

PMID: 12455973

Similar Datasets

Project description:Cryptococcus neoformans, an opportunistic yeast pathogen, relies on a complex network of stress response pathways that allow for proliferation in the host. In Saccharomyces cerevisiae, stress responses are regulated by integral membrane proteins containing a transient receptor potential (TRP) domain, including the flavin carrier protein 1 (Flc1), which regulates calcium homeostasis and flavin transport. Here, we report that deletion of C. neoformans FLC1 results in cytosolic calcium elevation and increased nuclear content of calcineurin-dependent transcription factor Crz1, which is associated with an aberrant cell wall chitin overaccumulation observed in the flc1Δ mutant. Absence of Flc1 or inhibition of calcineurin with cyclosporine A prevents vacuolar fusion under conditions of combined osmotic and temperature stress, which is reversed in the flc1Δ mutant by the inhibition of TORC1 kinase with rapamycin. Flc1-deficient yeasts exhibit compromised vacuolar fusion under starvation conditions, including conditions that stimulate formation of carbohydrate capsule. Consequently, the flc1Δ mutant fails to proliferate under low nutrient conditions and displays a defect in capsule formation. Consistent with the previously uncharacterized role of Flc1 in vacuolar biogenesis, we find that Flc1 localizes to the vacuole. The flc1Δ mutant presents a survival defect in J774A.1 macrophage cell-line and profound virulence attenuation in both the Galleria mellonella and mouse pulmonary infection models, demonstrating that Flc1 is essential for pathogenicity. Thus, cryptococcal Flc1 functions in calcium homeostasis and links calcineurin and TOR signaling with vacuolar biogenesis to promote survival under conditions associated with vacuolar fusion required for this pathogen's fitness and virulence. IMPORTANCE Cryptococcosis is a highly lethal infection with limited drug choices, most of which are highly toxic or complicated by emerging antifungal resistance. There is a great need for new drug targets that are unique to the fungus. Here, we identify such a potential target, the Flc1 protein, which we show is crucial for C. neoformans stress response and virulence. Importantly, homologues of Flc1 exist in other fungal pathogens, such as Candida albicans and Aspergillus fumigatus, and are poorly conserved in humans, which could translate into wider spectrum therapy associated with minimal toxicity. Thus, Flc1 could be an "Achille's heel" of C. neoformans to be leveraged therapeutically in cryptococcosis and possibly other fungal infections.

Project description:The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the alpha mating type (MAT(alpha)). We characterized C. neoformans mating pheromones (MF(alpha) 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MF(alpha) 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MF(alpha) 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MAT(alpha) strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MF(alpha) 1 identical to that of C. neoformans var. grubii. MF(alpha) 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MF(alpha) 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These observations could form the basis for future studies on the role of intervariety mating in C. neoformans biology and virulence.

Dataset Information

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

Publications

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets