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Background: Microsporum canis is a pathogenic dermatophyte that causes a superficial cutaneous mycosis, mainly in cats and humans. Proteolytic enzymes, including subtilisins, have been postulated to be key factors involved in adherence and invasion of the stratum corneum and keratinized epidermal structures., Objectives: To evaluate the importance of Sub3 as a M. canis virulence factor using a SUB3 RNA-silenced strain., Materials and Methods: The stability of a previously constructed RNA-silenced strain IHEM 22957 was tested in three different ways. The involvement of Sub3 in the adherence process was evaluated using a new ex vivo adherence model of M. canis arthroconidia to feline epidermis. In order to investigate the contribution of Sub3 in epidermal invasion, the pathogenicity of the SUB3 silenced strain was compared with that of the control strain in a guinea pig model of experimental M. canis dermatophytosis., Results: The silenced strain was shown to be stable after four in vitro transfers and after the in vivo experimental infection. This strain has dramatic loss of adherence capacity to feline corneocytes when compared with the parental strain. In contrast, no significant differences were observed at any time during the infection between the control strain and the SUB3 silenced strain, indicating that Sub3 secretion is not required for invasion of epidermal structures., Conclusions: RNA interference is a useful tool to evaluate pathogenic mechanisms of M. canis. For the first time, a role in pathogenicity could be attributed to a protease of a dermatophyte, namely Sub3 from M. canis, which is required for adherence to but not for invasion of the epidermis.