Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

Abstract

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

“…No fewer than 16 genes encoding secreted endo-and exopeptidases have been found in dermatophytes (Jousson et al, 2004a, b;Monod et al, 2005). The hypothesis that one keratinase, alone or with further proteases, decomposes hard keratin, has now been abandoned (Kunert, 1992).…”

“…For amplification, a standard protocol was used (Jousson et al, 2004a, b). Expression plasmids were constructed by cloning PCR products in the S. cerevisiae expression vector pYES2.…”

“…PCR was performed with the homologous primers P1-P6 (Table 1) and 200 ng of DNA prepared from 10 6 clones of the cDNA library, using a standard protocol (Jousson et al, 2004a, b).…”

“…Indeed, it is known that the nucleotide sequences of dermatophyte genes exhibit 50-70 % identity to the orthologous genes of Aspergillus spp. (Jousson et al, 2004a, b;Monod et al, 2005).…”

“…The battery of proteases secreted by dermatophytes is similar to that of Aspergillus, but differs by multiple endoprotease members of the S8 (subtilisins) and M36 (fungalysins) family (see MEROPS proteolytic enzyme database; http://merops.sanger. ac.uk) (Jousson et al, 2004a, b). Although it is evident that the secreted proteolytic activity of these fungi is important for degradation of compact keratinous tissues, fungal secreted proteases are incapable by themselves of dissolving structures made of hard keratin which are rich in cystine.…”