Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

Abstract

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo-and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection

“…Samples of A. benhamiae grown in either soy (soyA/B/C) or keratin-soy medium (keratin-soyA/B/C) were compared with probes derived from cells grown in control Sabouraud medium (SabA/B/C). The latter is known to induce less proteolytic activity by dermatophytes than media containing proteins as the sole source of carbon and nitrogen (Jousson et al, 2004a, b;Monod et al, 2005). We found 207 and 289 A. benhamiae sequences to be upand downregulated in soy medium, respectively (Supplementary Table S1A), and 221 and 216, respectively, in keratin-soy (Supplementary Table S1B) versus the control.…”

“…Similar to our previous analysis of protein degradation by the related dermatophyte T. rubrum, two commonly used protein media were tested in this approach, liquid soy medium and keratin-soy medium (Monod et al, 2005;Zaugg et al, 2009). In keratin-soy medium, the initial growth of the fungus on the insoluble keratin granules is facilitated by the addition of a small amount (0.01 %) of soy protein.…”

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes

“…Sulphitolysis is so far the sole known dermatophyte mechanism that allows the reduction of disulphide bridges, a bottleneck in the process of degradation of compact keratinized tissues. In contrast, the activities of the dermatophyte-secreted proteases are redundant for the digestion of reduced protein (Jousson et al, 2004b;Monod et al, 2005). Therefore, sulphite transporters, rather than secreted proteases, could be a target of choice to treat dermatophyte infection.…”

“…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).…”

“…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).…”