Secreted subtilisins of Microsporum canis are involved in adhesion of arthroconidia to feline corneocytes

Microsporum canis is a pathogenic fungus that causes a superficial skin infection called dermatophytosis mainly in cats, dogs and humans. Like other dermatophytoses, the physiopathology of this dermatosis remains largely unknown. From a fungal perspective, the infection process can be divided in three steps: adhesion of M. canis arthroconidia to corneocytes, conidial germination, and fungal invasion of the keratin network. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. However, several previously characterized secreted fungal endoproteases like subtilisins (Sub), including the keratinolytic protease Sub3, are secreted in vivo and could be involved in the first pathogenic steps. The objective of this study were (1) to develop an in vitro model to study M. canis adherence to feline corneocytes and (2) to assess whether the Sub are involved in fungal adhesion. An arthroconidial suspension was spread over the surface of reconstituted feline epidermis (RFE). Co-cultures were incubated for varying lengths of time and adherent conidia were labelled using Calcofluor white and counted. In subsequent assays arthroconidia were exposed to the serine protease inhibitor chymostatin or a mixture of two anti-Sub3 monoclonal antibodies (Mabs) one hour prior to the adherence assay. In our model, adherence of M. canis arthroconidia to RFE is time-dependent, beginning within two hours and still increasing after six hours. Chymostatin and Mabs inhibit M. canis adherence to RFE by 53 and 23 % respectively, which suggests that subtilisins and particularly Sub3, are fungal virulence factors involved in the adherence process.