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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.

SUBMITTER: Zuber S

PROVIDER: S-EPMC118015 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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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.

Zuber Sophie S Hynes Michael J MJ Andrianopoulos Alex A

Eukaryotic cell 20020601 3

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. T ...[more]

PMID: 12455992

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Project description:BackgroundPenicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37 degrees C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei.ResultsWhole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins.ConclusionThis study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.

Project description:BACKGROUND: Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus. METHODOLOGY/PRINCIPAL FINDINGS: We attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 candidates in mycelial and seven in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 among the thermal dimorphic fungi. Moreover, dcl-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1(KO), dcl-2(KO), dcl(DKO) and qde-2(KO) deletion mutants, we showed that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. The mRNA expression levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1 (KD), supporting regulatory function of milRNAs. CONCLUSIONS/SIGNIFICANCE: Our findings provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism.

Project description:During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host's defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells.

Dataset Information

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.

Publications

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.

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