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Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton
1Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Abstract:
Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.
Insights
Researchers cloned and characterized the first 70kDa heat-shock protein (HSP) from Trichophyton rubrum, a common cause of chronic fungal infections. This HSP is constitutively expressed and up-regulated by heat stress.
Area of Science:
- Medical Mycology
- Molecular Biology
- Protein Biochemistry
Background:
- Trichophyton rubrum causes most chronic dermatophytosis.
- Understanding pathogen proteins is crucial for molecular characterization.
- Heat-shock proteins (HSPs) are vital cellular components.
Purpose of the Study:
- To clone and characterize the 70kDa heat-shock protein (HSP70) gene from Trichophyton rubrum.
- To analyze the expression patterns of the identified HSP70 gene.
- To determine the HSP70 gene family size in T. rubrum.
Main Methods:
- cDNA library construction from T. rubrum.
- Recombinant protein expression and detection via Western blot.
- Genomic DNA analysis using Southern blot.
- RNA expression analysis via Northern blot.
Main Results:
- A recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs) was identified.
- Southern blot analysis indicated a single HSP70 gene family member in T. rubrum.
- The HSP70 gene contains two introns.
- Northern blot revealed constitutive expression at 27°C, with strong upregulation at 37°C.
Conclusions:
- The first HSP gene from a dermatophyte, T. rubrum, was cloned and identified as a member of the DnaK subfamily of 70kDa HSPs.
- The characterized HSP70 exhibits heat-inducible expression, suggesting a role in stress response.
- This study provides a molecular basis for further investigation into T. rubrum pathogenesis and stress adaptation.