Is the Difference in Keratinase Activity of Dermatophytes to Different Keratinaceous Substrates an Attribute of Adaptation to Parasitism?

Dermatophytes can digest keratin and other proteinaceous substrates present in skin and its appendages such as nail, hair, and feather and use it as its sole source of carbon and nitrogen. Proteolytic and keratinolytic activities of dermatophytes have been a subject of interest for several years to understand the pathogenicity of infection. In this study we intend to elucidate the keratinase activity profile among the three ecological groups viz. geophilic, zoophilic and anthrophophilic of dermatophytes. Six isolates of each species of T. rubrum and E. floccosum, M. gypseum and M. canis were grown on mineral medium consist of human hair, human nail and chicken feather individually. The keratinolytic activity was measured spectrophotometrically at 660 nm following Folin-Ciocalteu method. The data collected was analyzed using ANOVA followed Duncan Multiple Range test. A statistically significant difference (P= 0.000**) was noted in the enzyme activity of different ecological groups of dermatophytes. T. rubrum and E. floccosum (anthrophophilic deramtophytes) recorded the lowest activity for all the substrates when compared to the geophilic (M. gypesum) and zoophilic (M. canis) counterparts. We presume that the enzyme moderation could be an attribute for obligate Anthropization in certain dermatophytes. Egyptian Dermatology Online Journal Vol. 6 No 1: 6, June 2010 2 http://www.edoj.org.eg Introduction Keratins are the most abundant proteins in epithelial cells of vertebrates and represent the major constituent of skin and its appendages such as nail, hair, feather and wool. Keratin is a stable insoluble protein with rigid structure due to the presence of several cross-linking disulfide bonds. Dermatophytes can digest keratin and other proteinaceous substrates present in the skin, nail, hair etc. and use it as its sole source of carbon and nitrogen [1]. Proteolytic and keratinolytic activities of dermatophytes have been a subject of interest for several years to understand the pathogenicity of infection [2]. Dermatophytes of different ecological groups have evolved owing to their differences in host specificity. However a detailed comparative study of the selective preference of the different native keratin substrates among the ecological groups of dermatophytes and its possible co-relation to pathogenicity is lacking. Therefore in the current study we intended to elucidate the difference in the keratinase activity of Trichophyton rubrum & Epidermophyton floccosum (Anthrophophilic) Microsporum gypseum (Geophilic) and Microsporum canis (Zoophilic) on keratin substrates such as human hair, human nail and chicken feather. Materials And Methods Six isolates each of T. rubrum and E. floccosum (clinical origin), M. gypseum (2 clinical, 4 soil origin) and M. canis (from pet dogs) grown on Sabouraud Dextrose broth were used in the current study. Twenty micro liters of the fungal spore suspension of the dermatophytes viz. T. rubrum, E. floccosum, M. of MgSO4.7H2O, 0.1g of Cacl2.2H2O, pH 7.5 gypseum, and M. canis prepared in distilled water and adjusted to an absorbance of 0.6 at 450 nm were used as inoculums for the present study. Nine sets of 10 ml mineral medium (g/l of 0.5g KCl, 1.0g K2HPO4, 0.5g MgSO4, 2.0g NaNO3, 0.01g FeSO4.7H2O, 30g Dextrose) were prepared in 25 ml conical flasks containing 1% of different native keratin substrates viz. human hair, human nail and chicken feather individually for each isolate of each species. The above substrates viz. human hair, human nail and chicken feather were washed with ethanol, dried, and hammer milled individually prior to addition to the medium. The inoculated conical flasks were incubated on a rotary shaker (130rpm) at room temperature. From 4th day of incubation one flask (1/9) per strain per substrate was taken for keratinase enzyme assay. Keratinase assay was done on every alternate day from 4th day of incubation till 20 day [3]. One and half ml of culture was transferred to Eppendorf tube and centrifuged at 5000 rpm for 10 minutes. The culture supernatant (crude enzyme extract) was used for enzyme assay. A known volume (500ul) of the supernatant was incubated with 1% (w/v) keratin powder (Hi-media, India) in 20 mM Tris-HCl buffer (pH 8.0) at 30°C for 5h. The reaction was terminated with 6% (w/v) Tri Chloro Acetic acid (TCA) and then allowed to stand for 30min followed by centrifugation at 15,000xg for 10min. The resultant supernatant was mixed with 1:1 dilution of Folin-Ciocalteu reagent and 0.5 M NaOH and then incubated at room temperature for 1hour. The keratinolytic activity was measured spectrophotometrically (PerkinElmer) at 660 nm. The keratinolytic Egyptian Dermatology Online Journal Vol. 6 No 1: 6, June 2010 3 http://www.edoj.org.eg activity was expressed in keratin units (KU), and 1KU is defined as an increase of 0.01 OD at 660 nm in 1h [3,4]. The data collected was analyzed using ANOVA followed Duncan Multiple Range test.