Metalloprotease Gene Family in Microsporum canis Identified as Potential Virulence Factors Original paper

What was studied?

This study characterized a family of secreted metalloprotease genes in Microsporum canis, a zoophilic dermatophyte responsible for dermatophytosis in cats, dogs, and humans. The authors identified three homologous genes—MEP1, MEP2, and MEP3—encoding zinc-dependent metalloproteases containing the canonical HEXXH motif. These genes were isolated using a genomic probe derived from the Aspergillus fumigatus MEP gene. Among these, MEP3 was confirmed to encode the previously described 43.5-kDa keratinolytic metalloprotease and was successfully expressed in Pichia pastoris as a recombinant enzyme. To assess potential involvement in pathogenesis, the in vivo expression of MEP1–3 during infection was evaluated using RT-nested PCR on RNA extracted from infected guinea pig hair samples.

Who was studied?

The experimental infection model involved two 3-month-old female guinea pigs (Hartley strain) infected with M. canis strain IHEM 15221. Hair samples were harvested from infected and control animals at 14 and 21 days post-infection for molecular analysis of MEP gene expression. Additionally, P. pastoris was employed as a heterologous expression system to produce recombinant MEP3 for biochemical characterization.

Most important findings

The study provided the first genomic and functional characterization of a metalloprotease gene family in Microsporum canis, implicating these enzymes in fungal virulence. All three identified MEP genes—MEP1, MEP2, and MEP3—encode zinc-dependent metalloproteases belonging to the M36 (fungalysin) family. Among them, MEP3 demonstrated broad proteolytic activity, including keratinolytic, elastinolytic, and collagenolytic functions, distinguishing it from its Aspergillus homologs, which cleave only collagen. In vivo expression analysis via RT-nested PCR confirmed that MEP2 and MEP3, but not MEP1, are transcribed in infected hair samples during the phase of lesion development. MEP3 was further expressed in Pichia pastoris as a recombinant enzyme, yielding approximately 40 µg/mL and retaining native proteolytic activity. N-terminal sequencing of the recombinant protein matched the native MEP3, validating accurate folding and post-translational processing in the heterologous system.

Key implications

This work demonstrates that M. canis expresses multiple metalloproteases during infection, with MEP3 likely contributing to keratin degradation and host tissue invasion. Given the evidence for its in vivo expression and enzymatic activity, MEP3 emerges as a strong candidate virulence factor and a potential immunogen for vaccine development. The absence of MEP1 expression in vivo suggests a temporally or spatially restricted role or low expression levels. The identification of this gene family lays the groundwork for functional genetic studies, including gene knockouts to verify roles in pathogenicity, and may facilitate the development of diagnostic or therapeutic tools targeting metalloproteases in zoonotic dermatophytosis.