Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

enzymatic activity and molecular characterization of a secreted subtilisin-like protease in Microsporum gypseum and Trichophyton vanbreuseghemii

Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which degraded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study. Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like proteases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR. The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermatophytes. Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes

Keratinolytic activity of five human isolates of the dermatophytes.

The keratinolytic activity of five species of the dermatophytes which include Trichophyton rubrum, T. mentagrophytes, T. tonsurans, Microsporum audouinii and M. gypseum isolated from school children were tested using human hair as the substrate. M. gypseum was found to possess the highest keratinolytic activity with a net value of released protein being 78.8 ug/ml after five weeks of incubation. Also the net value of released protein for T. tonsurans, T. rubrum, T. mentagrophytes and M. audouinii were 55.5 ug/ml, 52.5 ug/ml, 43.8 ug/ml and 26.3 ug/\ml respectively. Only T. mentagrophytes and M. gypseum were able to cause structural damage in form of perforations on the hair shaft. Also during the degradation of the hair, the pH of the basal medium for each dermatophyte increased. The increase in pH was highest in the medium with M. gypseum but lowest in that of M. audouinii.

Isolation, Purification and Characterization of Keratinolytic Proteinase from Microsporum canis

A keratinolytic proteinase secreted by Microsporum canis in a broth containing human hair was purified 134-fold from the culture filtrate by ion-exchange chromatography using DEAE-Sephacel, CM-Sephadex C-50, and by Sephadex G-75 gel filtration. The purified enzyme was electrophoretically homogeneous with a molecular weight of 33,000. The enzyme had an optimum pH of 8.0, and the activity was stable in the alkaline pH range. Enzyme activity increased with temperature up to 35 degrees C and was stable up to 45 degrees C. The keratinolytic activity was not affected by the addition of nonionic detergents, was activated by Mg2+, but inhibited by Zn2+. The purified enzyme was used to obtain guinea pig antiserum. The antiserum tested by double diffusion against the purified enzyme showed a single line of precipitation and completely neutralized the proteinase activity. This study reaffirms that the proteinase from M. canis may be a biochemical mechanism for the invasion of keratinized tissue, and could possibly play a role in the hypersensitivity reactions arising from superficial infections of this fungus.