Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.     

The First Isolation of Arthroderma benhamiae in Japan

A clinical isolate of Trichophyton mentagrophytes from rabbit was examined by polymerase chain reaction (PCR) analysis and a mating experiment. The species-specific primers designed from the nucleotide sequences of the chitin synthase 1 (CHS1) gene in the teleomorph of Arthroderma benhamiae amplified a fragment from genomic DNA samples of A. benhamiae and the clinical isolate but not from those of A. simii and A. vanbreuseghemii. On the other hand, the species-specific primers of A. simii and A. vanbreuseghemii did not amplify any fragment from the genomic DNA of the clinical isolates. When the isolate was respectively crossed with (+) or (-) tester strains of A. benhamiae, A. simii and A. vanbreuseghemii, ascospores were produced in the crossing with the A. benhamiae (+) strain. Therefore, the isolate was identified to be A. benhamiae (-), confirming the result of molecular analysis. This is the first report on the isolation of A. benhamiae in Japan.     

Cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otae-related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae-related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1-homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1-homologous group of M. gypseum complex are more diverse. This ITS1-based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time-saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non-culturable state.