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Cloning and characterization of bys1, a temperature-dependent cDNA specific to the yeast phase of the pathogenic dimorphic fungus Blastomyces dermatitidis.

ABSTRACT: The pathogenic dimorphic fungal organism Blastomyces dermatitidis exists as a budding yeast at 37 degrees C and as a mycelium at 25 degrees C. While the conversion of one morphological phase of B. dermatitidis to another has long been known to be a thermally dependent process, little of the accompanying biochemical or genetic events controlling the phase transition has been elucidated. Using differential cDNA library screening, we have identified one transcript, bys1, in B. dermatitidis that is expressed at very high levels in the yeast phase but whose levels diminish rapidly when yeast cells are transferred to 25 degrees C to promote conversion to the mycelial phase. Although the 0.95-kb bys1 transcript is absent in B. dermatitidis mycelia maintained at 25 degrees C, transfer of mycelial cultures to 37 degrees C results in the reappearance of bys1 within 12 h. bys1 codes for a protein of 18.6 kDa that contains multiple putative phosphorylation sites, a hydrophobic N terminus, and two 34-amino-acid domains with similarly spaced nine-amino-acid degenerative repeating motifs. Although the nature of the thermal dependency of bys1 expression and the function of the bys1 protein are unknown, the strong expression of this transcript specifically in the yeast phase of B. dermatitidis may prove to be very useful in the development of more specific and sensitive diagnostic methods for blastomycosis.

SUBMITTER: Burg EF

PROVIDER: S-EPMC186540 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

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Cloning and characterization of bys1, a temperature-dependent cDNA specific to the yeast phase of the pathogenic dimorphic fungus Blastomyces dermatitidis.

Burg E F EF Smith L H LH

Infection and immunity 19940601 6

The pathogenic dimorphic fungal organism Blastomyces dermatitidis exists as a budding yeast at 37 degrees C and as a mycelium at 25 degrees C. While the conversion of one morphological phase of B. dermatitidis to another has long been known to be a thermally dependent process, little of the accompanying biochemical or genetic events controlling the phase transition has been elucidated. Using differential cDNA library screening, we have identified one transcript, bys1, in B. dermatitidis that is ...[more]

PMID: 8188377

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Project description:Genes expressed in the parasitic yeast (Y) phase of the dimorphic fungal pathogen Histoplasma capsulatum which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To understand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5' regulatory regions of two Y phase-specific genes (yps-3 and yps 21:E-9) were PCR amplified as labelled probes to identify nuclear DNA binding proteins which may influence phase-specific gene transcription. Protein-DNA interactions were assessed by Southwestern blot analysis in which sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated protein extracts from Y and M phases of the virulent G217B strain of H. capsulatum were visualized by their capability for in situ binding to the labelled 517-bp (G217B yps-3) or the 395-bp (G217B yps 21:E-9) putative 5' regulatory regions. A 30-kDa nuclear protein unique to the M-phase extracts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, thermal-sensitive Downs strain of H. capsulatum lacked detectable p30 binding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B yps 21:E-9 probe or 512-bp HindIII-EcoRI-labelled Downs yps21:E-9). A decanucleotide motif, TCCTTTTTTT, was identified in the upstream regulatory regions of these yps genes, as well as in the putative alpha-tubulin promoter, and was conserved with 70 to 100% homology. This recognition sequence was sufficient for p30M binding with 32P-labelled ligated oligonucleotides when used in the Southwestern assay. These findings describe the first nuclear DNA binding factor identified in H. capsulatum which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in which a temperature-sensitive switch influences morphology and virulence.

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.

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Dataset Information

Cloning and characterization of bys1, a temperature-dependent cDNA specific to the yeast phase of the pathogenic dimorphic fungus Blastomyces dermatitidis.

Publications

Cloning and characterization of bys1, a temperature-dependent cDNA specific to the yeast phase of the pathogenic dimorphic fungus Blastomyces dermatitidis.

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