Isolation of a Microsporum canis Gene Family Encoding Three Subtilisin-Like Proteases Expressed in vivo

Abstract

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Allergy and Dermatophytes

“…In support of this, computer-assisted molecular modeling of Tri r 2 predicted the same three-dimensional structure as the structurally conserved regions of another subtilase, proteinase K, which exhibits high proteolytic activity (124). Identification of a gene family encoding three subtilisin-like proteinases derived from M. canis has recently been described (24). One of these genes encoded a keratinase that was previously isolated (79).…”

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

“…The present study was performed on T. rubrum, the most frequent dermatophyte found in man in European countries (Monod et al, 2002). We have shown herein that, in addition to endoproteases of the S8 and M36 families, T. rubrum secretes leucine aminopeptidases (Lap) of the M28 family, and dipeptidyl-peptidases (Dpp) of the S10 family.…”

“…The nucleotide sequences of dermatophyte endoprotease genes (Descamps et al, 2002;Brouta et al, 2002;Jousson et al, 2004a, b) exhibited 50-70 % identity to homologous genes in Aspergillus spp. Therefore, we investigated the aminopeptidase activity of T. rubrum by a reverse genetic approach (from genes to proteins).…”

“…Recent investigations have shown that proteases secreted by dermatophytes are similar to those of other fungi such as Aspergillus spp. (Brouta et al, 2002;Descamps et al, 2002;Jousson et al, 2004a, b), and are members of large protein families. In particular, two gene families encoding endoproteases of the S8 (subtilisins) and M36 (fungalysins) families (see the MEROPS proteolytic enzyme database, http://merops.sanger.ac.uk/) were found in Trichophyton rubrum, Trichophyton mentagrophytes, Arthroderma benhamiae and Microsporum canis.…”

Comparative analysis of secreted enzymes between the anthropophilic–zoophilic sister species Trichophyton tonsurans and Trichophyton equinum

“…Facilitating the infection process is a complement of secreted enzymes that function collectively to provide a pathway for fungal invasion and liberate nutrients from the keratin-rich structures that comprise the outer integument of the animals that they infect (Apodaca & McKerrow 1989a, b;Weitzman & Summerbell 1995;Monod et al 2002Monod et al , 2005Jousson et al 2004b;Kano et al 2005;Giddey et al 2007a;Vermout et al 2008;Zaugg et al 2009). While these enzymes appear to be conserved among the dermatophytes examined to date (Apodaca & McKerrow 1989a;Mignon et al 1998;Brouta et al 2001Brouta et al , 2002Descamps et al 2002;Jousson et al 2004a, b;Bhathena et al 2005;Kano et al 2005;Monod et al 2005;Moallaei et al 2006;Giddey et al 2007a, b;Vermout et al 2008), the manner in which they are expressed appears to be relatively species-specific (Giddey et al 2007a). Notably, this holds for dermatophytes that are genetically very closely related (Graser et al 1999;Summerbell et al 1999).…”