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

The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium

In this communication, we show that the growth of isolate H6 of the dermatophyte Trichophyton rubrum on non-buffered medium and under saturating phosphate conditions is dependent on the initial growth pH, with an apparent optimum at pH 4.0. In addition, irrespective of the initial growth pH, the pH of the medium alteredduring cultivation reaching values that ranged from 8.3 to 8.9. Furthermore, this isolate synthesized and secreted almost the same levels of an alkaline phosphatase with an apparent optimum pH ranging from 9.0 to 10.0 when grown on both low- and high-phosphate medium. Also, this alkaline phosphatase is activated by Mg2+ and is EDTA-sensitive. On the other hand, the very low levels of the enzyme retained by the mycelium grown on buffered medium at pH 5.0-5.2 suggest that this enzyme is encoded by an alkaline gene, i.e., a gene responsive to ambient pH signaling.

Inhibitory effect of aqueous onion and garlic extracts on growth and keratinase activity in trichophyton mentagrophytes

The effect of onion and garlic extracts on fungal growth and keratinolytic activity was studied in Trichophyton mentagrophytes as one of the major etiologic agents of human and animal dermatophytosis in Iran and other parts of the world. In order to find out the best keratinase producer for further steps, culture conditions for 30 strains of T. mentagrophytes isolated from human dermatophytosis were optimized on specific solid and liquid media. All of the isolates produced the enzyme on both selective culture media. The maximum keratinolytic activity at submerged cultivation was reported for cultures of T. mentagrophytes isolate No. 1 grown for a 12-day period at 32°C. Extracellular keratinase activity was in the range of 0.28 to 2.18 u/mg protein in different isolates at predetermined optimal conditions. The growth of T. mentagrophytes isolate No. 1 was inhibited in the presence of various concentrations of onion and garlic extracts. This inhibition reached to a maximum of 100% for both extracts at 10% v/v concentrations. Keratinase synthesis was also inhibited by two extracts as a dose-dependent manner with maximums about 58.54 and 71.36% at 5% concentrations, accordingly. In contrast to the fungal growth, keratinolytic activity was inhibited more by garlic as compared with onion extract. This is the first report on keratinase inhibition by these two natural compounds. Since fungal growth and keratinolytic activity are important factors in pathogenesis of the dermatophytes, their inhibition by onion and garlic indicate that these substances may have potential values for treatment of human and animal dermatophytosis

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.

Relationship between Candida albicans producing proteinase (CAPP) and its environmental pH--comparison with a case of trichophyton mentagrophytes

Candida albicans produced a karatinolytic proteinase (KPase) or C. albicans producing proteinase (CAPP), a proposed new term for this enzyme, and Trichophyton mentagrophytes also produced KPase when cultivated in liquid medium containing human stratum corneum (HSC) as the nitrogen source, but were unable to do so when cultivated in sabouraud dextrose broth. Purified KPase from the culture supernatants of C. albicans had a molecular weight of 42,000 and an optimum pH at 4.0. The KPase was found to belong to the carboxyl proteinases group and its activity was strongly inhibited by pepstatin. Both fungi were able to grow by secreting KPase which digested HSC for nutrients. KPase from both fungi had high activity in each optimum pH, such as weakly acidic pH on C. albicans and neutral pH on T. mentagrophytes to adapt their surrounding environment by changing the environmental pH into their own optimum pH.