Please cite this article as: Bȃguţ, E.T., Baldo, A., Mathy, A., Cambier, L., Antoine, N., Cozma, V., Mignon, B., Sub3 is involved in adherence of Microsporum canis to human and animal epidermis, Veterinary Microbiology (2010Microbiology ( ), doi:10.1016Microbiology ( /j.vetmic.2012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The aim of this study was to assess the role of the secreted keratinolytic subtilisin-like Miracloth layers (22-25 µm; Calbiochem, La Jolla, CA, USA) and stored at 4 °C until use.
87Arthroconidia concentration was determined by serial dilutions on Sabouraud's agar medium.
88In all the experiments, arthroconidia were used within two weeks. A c c e p t e d M a n u s c r i p t for adherence to feline epidermis. Three independent assays using skin explants from three 151 unrelated cats were performed as described above, except that before inoculation of skin 152 explants with deficient or control strain arthroconidia, the latter were preincubated with rSub3
153(2.5 µg/ml, 1 µg/ml or 0.5 µg/ml reaction mixture) for 1 h at 37 °C in humidified atmosphere 154 containing 5% CO 2 . Recombinant Sub3 was substituted with PBS as a control.
155Finally, the stability of rSub3 in the conditions of our adherence assays, i.e., after contact with 156 either feline epidermis or arthroconidia, was also assessed. The rSub3 was either deposited on 157 feline epidermis or mixed with deficient or control strain arthroconidia for 4 h at 37 °C.
158Samples were then subjected to 12% sodium dodecyl sulfate polyacrylamide gel 159 electrophoresis (SDS-PAGE) under reducing conditions and assessed for enzymatic activities 160 using Suc-AAPF-pNa, as described above. arthroconidia from the M. canis SUB3 silenced strain were not stained (Fig. 1c). As a control incubated with pre-immune antibody (Fig. 1b) epidermis when compared to the control strain set up as 100% (Fig. 2). These results show which its expression has also been proven.
229For dermatophytes, the precise mechanisms by which subtilisins contribute to virulence are on the adherence of either silenced or control strain arthroconidia (Fig. 3). The absence of 250 adherence restoration upon addition of rSub3 was not related to degradation of exogenous 251 rSub3 during adherence assays. Indeed, after contact with either feline epidermis (Fig. 4)
