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Identification and characterization of a phase-specific, nuclear DNA binding protein from the dimorphic pathogenic
1Center for Molecular Studies, J. C. Self Research Institute, Greenwood Genetics Center, Greenwood, South Carolina 29646, USA.
Abstract:
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.
Insights
Researchers identified a unique 30-kDa nuclear protein (p30M) in the mycelial phase of Histoplasma capsulatum. This protein binds to specific DNA sequences, suggesting it regulates gene transcription and influences the fungus's virulence and morphology.
Area of Science:
- Mycology
- Molecular Biology
- Pathogenesis
Background:
- Dimorphic fungi like Histoplasma capsulatum switch between yeast (Y) and mycelial (M) phases, impacting virulence.
- Understanding the molecular mechanisms regulating this phase transition is crucial for controlling fungal infections.
Purpose of the Study:
- To identify nuclear DNA-binding proteins involved in the phase-specific gene regulation of Histoplasma capsulatum.
- To elucidate the role of these proteins in the transition and maintenance of the yeast phase.
Main Methods:
- PCR amplification of 5' regulatory regions of Y phase-specific genes (yps-3, yps 21:E-9) as labeled probes.
- Southwestern blot analysis using protein extracts from Y and M phases of virulent (G217B) and low-virulence (Downs) H. capsulatum strains.
- Identification of a conserved decanucleotide motif (TCCTTTTTTT) in regulatory regions.
Main Results:
- A 30-kDa nuclear protein (p30M) was identified, unique to the M phase of the virulent G217B strain and absent in the Y phase.
- The low-virulence Downs strain showed no detectable p30 binding activity.
- The decanucleotide motif TCCTTTTTTT was found in yps gene promoters and the alpha-tubulin promoter, and was sufficient for p30M binding.
Conclusions:
- p30M is the first identified nuclear DNA-binding factor in H. capsulatum exhibiting phase-specific binding.
- p30M likely plays a significant role in regulating gene transcription during the mycelial phase.
- These findings provide insights into the molecular control of morphology and virulence in this pathogenic fungus.