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Molecular cloning and sequence analysis of yps-3, a yeast-phase-specific gene in the dimorphic fungal pathogen
Abstract:
Genes specifically expressed in the parasitic yeast phase of Histoplasma capsulatum have been cloned to clarify the mechanisms underlying both pathogenesis and morphogenesis in this human dimorphic fungal pathogen. Previous studies have determined that the yeast-phase-specific gene, yps-3, is expressed differentially in two non-isogenic strains which differ in their thermotolerance and virulence. We have cloned the yps-3 homologues from the high virulence (G217B) and low virulence (Downs) strains, and obtained a partial cDNA clone representing the expressed gene from H. capsulatum G217B. The Downs clone harbours a 287 bp insertion sequence that disrupts a long ORF defined by the yps-3 G217B cDNA. Although the insertion sequence contains features reminiscent of mobile genetic elements, including 15 bp direct repeats of flanking sequence, it is not randomly distributed in the H. capsulatum genome. S1 nuclease analysis was utilized to map the 5' end of the expressed yps-3 gene in G217B to potential regulatory regions which are largely homologous in both strains. This finding may point to a deficiency in a temperature inducible regulatory protein in the low virulence, temperature-sensitive Downs strain. The nucleotide sequence of the yps-3 gene and the predicted amino acid sequence of its product represents the first report of phase-specific gene and protein sequences in this widely distributed fungal pathogen. Further analysis of the product encoded by the yps-3 gene may provide significant insight into the pathogenic repertoire of H. capsulatum.
Insights
Researchers cloned the yeast-phase-specific gene yps-3 from Histoplasma capsulatum, revealing sequence differences linked to virulence. This study provides the first phase-specific gene and protein sequences for this fungal pathogen.
Area of Science:
- Medical Mycology
- Molecular Biology
- Genetics
Background:
- Histoplasma capsulatum is a dimorphic fungal pathogen causing histoplasmosis.
- Yeast-phase-specific genes are crucial for understanding H. capsulatum pathogenesis and morphogenesis.
- The yps-3 gene shows differential expression between high and low virulence strains.
Purpose of the Study:
- To clone and characterize yps-3 homologues from high (G217B) and low (Downs) virulence H. capsulatum strains.
- To investigate the molecular basis for differential gene expression and its relation to virulence.
- To report the first phase-specific gene and protein sequences in H. capsulatum.
Main Methods:
- Gene cloning of yps-3 homologues from G217B and Downs strains.
- cDNA cloning and sequencing.
- S1 nuclease analysis to map gene expression.
- Bioinformatic analysis of nucleotide and predicted amino acid sequences.
Main Results:
- The Downs strain yps-3 homologue contains a 287 bp insertion disrupting the open reading frame (ORF).
- The insertion sequence shares features with mobile genetic elements but is not randomly distributed.
- S1 nuclease analysis mapped the 5' end of the expressed yps-3 gene in G217B to conserved regulatory regions.
- This suggests a potential deficiency in a temperature-inducible regulatory protein in the Downs strain.
Conclusions:
- The study reports the first nucleotide and predicted amino acid sequences for a phase-specific gene (yps-3) in H. capsulatum.
- Sequence variations, including the insertion in the Downs strain, likely contribute to differences in thermotolerance and virulence.
- Further analysis of the yps-3 gene product may elucidate pathogenic mechanisms of H. capsulatum.