Targeted gene deletion and in vivo analysis of putative virulence gene function in the pathogenic dermatophyte
Maria Grumbt1, Valérie Defaweux, Bernard Mignon
1Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Junior Research Group Fundamental Molecular Biology of Pathogenic Fungi, Beutenbergstr. 11a, D-07745 Jena, Germany.
Abstract:
Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.
Insights
Malate synthase is essential for dermatophyte growth on lipids but not required for virulence in guinea pig skin infections. This study establishes a genetic system for Arthroderma benhamiae, aiding future research on fungal pathogenicity.
Area of Science:
- Medical Mycology
- Molecular Biology
- Fungal Pathogenesis
Background:
- Dermatophytes cause most human and animal superficial mycoses, but their pathogenicity mechanisms remain poorly understood.
- Molecular research on dermatophytes, including Arthroderma benhamiae, has been limited.
- The glyoxylate cycle enzyme malate synthase (AcuE) was recently found to be activated during A. benhamiae infections.
Purpose of the Study:
- To investigate the role of malate synthase (AcuE) in the pathogenicity of Arthroderma benhamiae.
- To establish and utilize a genetic system for targeted gene manipulation in A. benhamiae.
- To assess the contribution of AcuE to fungal growth on lipids and virulence in experimental models.
Main Methods:
- Development of the first genetic system for Arthroderma benhamiae, including gene deletion mutants (ΔacuE).
- Construction of mutants in both wild-type and KU70-deleted strains to enhance homologous recombination efficiency.
- Phenotypic analysis of mutants in vitro (growth on lipids) and in vivo (guinea pig infection model, reconstituted human epidermis model).
Main Results:
- Malate synthase (AcuE) is crucial for the growth of A. benhamiae on lipids, a key component of skin.
- Deletion of the KU70 gene facilitated targeted DNA insertion via homologous recombination without affecting growth or virulence.
- ΔacuE mutants did not show impaired virulence in guinea pig dermatophytosis or epidermal invasion models.
Conclusions:
- Malate synthase is essential for utilizing lipids but dispensable for Arthroderma benhamiae virulence in the studied models.
- The established genetic system for A. benhamiae is a valuable tool for characterizing fungal virulence factors.
- Further research is needed to fully understand the pathogenicity of dermatophytes.
