Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo
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Frédéric Descamps1, Frédéric Brouta, Michel Monod
1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Belgium.
Abstract:
Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.
Insights
Microsporum canis, a common cause of fungal infections in pets, secretes keratinolytic proteases. Researchers identified a family of these protease genes (SUB1, SUB2, SUB3), suggesting their role in causing dermatophytosis.
Area of Science:
- Mycology
- Molecular Biology
- Veterinary Dermatology
Background:
- Dermatophytosis in dogs and cats, caused by Microsporum canis, is a significant zoonotic disease.
- The pathogenesis of dermatophytosis is poorly understood, with fungal secreted proteases being potential virulence factors.
- Understanding these proteases is crucial for developing targeted treatments.
Purpose of the Study:
- To isolate and characterize genes encoding keratinolytic proteases secreted by Microsporum canis.
- To investigate the potential role of these proteases in the pathogenesis of dermatophytosis.
- To identify a gene family of potential virulence factors in dermatophytes.
Main Methods:
- Polymerase chain reaction (PCR) was used to obtain a gene fragment probe.
- A M. canis genomic library was screened using the probe to isolate protease genes.
- Gene sequencing and amino acid sequence analysis were performed.
- Reverse transcriptase nested-PCR was used to detect gene expression in infected animal hair.
Main Results:
- A 31.5 kDa keratinolytic subtilisin-like protease gene (SUB3) was cloned from M. canis.
- Two related genes (SUB1 and SUB2) were also identified.
- Sequence analysis revealed SUB1, SUB2, and SUB3 encode secreted proteases with homology to other fungal subtilisin-like proteases.
- mRNA for SUB1, SUB2, and SUB3 was detected in hair from experimentally infected guinea pigs.
Conclusions:
- SUB1, SUB2, and SUB3 constitute a gene family encoding subtilisin-like proteases in M. canis.
- These proteases are likely produced during M. canis invasion of keratinized tissues.
- This study reports the first isolation of a gene family encoding potential virulence factors in dermatophytes.
