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Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

ABSTRACT: In the dimorphism (mold/yeast) Histoplasma capsulatum (Hc) literature are reports that yeast (the so-called pathogenic form) uniquely expresses a cysteine dioxygenase (CDO, approx. 10,500 dal) activity which the mold morphotype (the so-called saprophytic soil form) does not express (C.F., Kumar et al., Biochem 22, 762, 1983). This yeast-specific CDO activity is postulated to play a critical role in the mold-to-yeast shift. A number of years ago, our lab isolated the gene encoding the Hc cysteine dioxygenase (CDO1, Genbank accession AY804144) and noted significant expression in the mold morphotype of several Histoplasma strains and also determined that the predicted protein would be over double the 10,500 dal reported by Kumar et al. Our report demonstrates (in the class 1 Downs strain, the class 2 G271B strain and two Panamanian strains, 184AS and 186AS) that the CDO1 gene is expressed in both the mold and yeast morphotypes and both morphotypes show significant CDO activity. Furthermore, we show via a FLAG-tag analysis that the expressed protein is approximately 24.7 ± 2.4 kd, in agreement with the putative protein sequence (determined from cDNA sequence) which yields 23.8 kd and is consistent with most other eukaryotic CDO enzymes. Additionally, we demonstrate that intracellular cysteine levels are actually significantly higher in the mold form of the two Panamanian strains, 184AS and 186AS, equal in both mold and yeast in the class 1 Downs strain and significantly higher in yeast of the more pathogenic class 2 G217B strain.

SUBMITTER: Adams MA

PROVIDER: S-EPMC7151203 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

Adams Melissa A MA Shearer Glenmore G

Journal of fungi (Basel, Switzerland) 20200213 1

In the dimorphism (mold/yeast) Histoplasma capsulatum (Hc) literature are reports that yeast (the so-called pathogenic form) uniquely expresses a cysteine dioxygenase (CDO, approx. 10,500 dal) activity which the mold morphotype (the so-called saprophytic soil form) does not express (C.F., Kumar et al., Biochem 22, 762, 1983). This yeast-specific CDO activity is postulated to play a critical role in the mold-to-yeast shift. A number of years ago, our lab isolated the gene enc ...[more]

PMID: 32069814

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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.

Dataset Information

Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus Histoplasma capsulatum Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

Publications

Cysteine Dioxygenase Enzyme Activity and Gene Expression in the Dimorphic Pathogenic Fungus <i>Histoplasma capsulatum</i> Is in both the Mold and Yeast Morphotypes and Exhibits Substantial Strain Variation.

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