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Isolation of transcripts over-expressed in human pathogen Trichophyton rubrum during growth in keratin
Fernanda C A Maranhão1, Fernanda G Paião, Nilce M Martinez-Rossi
1Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brazil.
Abstract:
Trichophyton rubrum is a cosmopolitan and anthropophilic fungus able to invade keratinized tissue, causing infection in human skin and nails. This work evaluated the changes in the extracellular pH during its growth in keratin (after 6, 12, 24, 48, 72h and 7 days) at initial pH 5.0. We observed a gradual increase of basal pH under keratin exposure when compared to glucose condition. Also, we identified 576T. rubrum transcripts differentially expressed by subtractive suppression hybridization (SSH) using conidia cultivated for 72h in keratin as tester, and cultivated in glucose as driver. The over-expression of 238 transcripts obtained under keratin condition was confirmed by macro-array dot-blot, revealing 28 unigenes. Putative proteins encoded by these genes showed similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisin and metalloprotease, cytochrome P450, GlcN-6-phosphate deaminase and Hsp30. The upregulation of T. rubrum genes encoding subtilisin, metalloprotease and Gag-Pol polyprotein was also validated by northern blot. The results of this study provide the first insight into genes differentially expressed during T. rubrum grown in keratin that may be involved in fungal pathogenesis.
Insights
Trichophyton rubrum causes skin and nail infections by invading keratin. This study identified key genes, including those for proteases and transporters, that are upregulated when the fungus grows on keratin, offering insights into its pathogenesis.
Area of Science:
- Medical Mycology
- Molecular Biology
- Biochemistry
Background:
- Trichophyton rubrum is a common cause of human superficial fungal infections, particularly affecting skin and nails.
- The fungus invades keratinized tissues, but the molecular mechanisms underlying this process are not fully understood.
Purpose of the Study:
- To investigate the differential gene expression of Trichophyton rubrum during growth on keratin.
- To identify specific genes and proteins involved in the fungal pathogenesis of keratinized tissues.
Main Methods:
- Cultivation of T. rubrum on keratin and glucose media, with extracellular pH monitoring.
- Subtractive suppression hybridization (SSH) to identify differentially expressed transcripts.
- Macro-array dot-blot and Northern blot to validate gene expression.
Main Results:
- Extracellular pH increased significantly during T. rubrum growth on keratin compared to glucose.
- SSH identified 576 differentially expressed transcripts, with 238 over-expressed on keratin.
- Validation confirmed upregulation of genes encoding subtilisin, metalloprotease, and Gag-Pol polyprotein, among others.
Conclusions:
- This study provides the first comprehensive analysis of genes differentially expressed by T. rubrum in a keratin-rich environment.
- Identified upregulated genes, including those encoding virulence factors like proteases, are likely crucial for fungal invasion and pathogenesis of skin and nail infections.
