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Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton
Gil Kaufman1,2, Benjamin A Horwitz2, Ruthi Hadar2
1Department of Molecular Microbiology, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 32000, Israel.
Abstract:
Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.
Insights
This study introduces a new method using green fluorescent protein (GFP) to visualize Trichophyton mentagrophytes during skin infections. This technique allows researchers to observe fungal growth and gene expression in real-time, advancing the study of dermatophyte pathogenicity.
Area of Science:
- Mycology
- Dermatology
- Molecular Biology
Background:
- Dermatophyte skin infections caused by Trichophyton are common.
- Investigating the molecular mechanisms of fungal pathogenicity is challenging due to limited research methods.
- Current methods like electron microscopy require tissue fixation, hindering dynamic observation.
Purpose of the Study:
- To develop a novel method for studying the molecular basis of Trichophyton pathogenicity.
- To enable real-time visualization of fungal growth and gene expression during skin infection.
- To establish a vital marker for observing dermatophyte-host interactions.
Main Methods:
- Utilized restriction-enzyme-mediated integration (REMI) for stable gene expression.
- Introduced green fluorescent protein (GFP) under a constitutive fungal promoter in Trichophyton mentagrophytes.
- Employed confocal microscopy to visualize fluorescent hyphae in culture and human skin explants.
Main Results:
- Achieved stable expression of GFP in a clinical isolate of Trichophyton mentagrophytes.
- Successfully visualized fluorescent fungal hyphae in both in vitro cultures and human skin models.
- Observed and documented stages of infection and penetration into skin layers.
Conclusions:
- REMI provides a robust method for generating genetically modified Trichophyton strains.
- GFP serves as an effective vital marker for studying dermatophyte infections in real-time.
- This approach offers unprecedented insights into fungal gene expression during host-pathogen interactions, advancing the understanding of skin infections.
