ABSTRACT
Dermatophytes infect the human hair, skin, nail and cause the dermatophytosis. The extracellular and intracellular proteinases of the dermatophytes commonly occur in the genus Trichophyton like T. rubrum, T. mentagrophytes, and T. granulosum. These enzymes play a prominent role in growth, multiplication and infection of the host tissue. Extracellular proteinases have been purified from the species of Trichophyton and Microsporum. We purified the proteinase partially from the culture filtrate of the Trichophyton tonsurans through Mono-Q and Superose 12 column and investigated its biochemical and enzymatic characters. The molecular size of the proteinase was estimated to be 41 kDa by SDS-PAGE. And pI was 3.2. The optimal temperature and pH for an enzymatic activity were 27C and 7.5, respectively. The purified porteinase degraded the keratin, bovine serum albumin, hemoglobin. The serine proteinase inhibitor like PMSF and DFP inhibited the proteolytic activity of the purified enzyme whereas the cysteinase inhibitor did not. These results demonstrated that the purified proteinase is a serine proteinase and can contribute the tissue invasion.
Subject(s)
Humans , Arthrodermataceae , Electrophoresis, Polyacrylamide Gel , Hair , Hydrogen-Ion Concentration , Isoflurophate , Microsporum , Peptide Hydrolases , Serine Proteases , Serine , Serum Albumin, Bovine , Skin , Tinea , TrichophytonABSTRACT
BACKGROUND: Dermatophytoses are infections of keratinized tissues, that is, the epidermis, hair and nails, caused by a group of specialized fungi, the dermatophytes. Laboratory diagnoses of dermatophytes such as Tricophyton, Microsporum and Epidermophyton are made by microscopic examination and in vitro culture but they are either time consuming of lacking specificity. OBJECTIVE: In order to develop and apply more rapid and precise diagnostic tests for fungal pathogens to facilitate the improved identification of dermatophytes, we investigated random amplified polymorphism DNA for classification and identification of dermatophytes. METHODS: Amplification reactions were performed in volumes of 5011 containing 10mM Tris-HCl(pH 8.3), 50mM KCl, 1.5mM MgCl2, 0.01% (w/v), gelatin, 200mM dNTP mixture, 50pM primer, Taq polymerase (0.025 units/ microliter), DNA 0.001 microgram/microliter. The optimal condition to. PCR was 2 cycles (denaturing 94 degrees C 2min, annealing 33 degrees C 2min, extension 72 degrees C 4min), 40 cycles, and extension (72 degrees C 10min). RESULTS: RAPD showed interspecies polymorphism in but it had identical patterns in intraspecies. CONCLUSION: It was confirmed that RAPD PCR analysis with optimal conditions is a fast, economical and reproducible method for identification and classification of dermatophytes isolates.
Subject(s)
Arthrodermataceae , Classification , Clinical Laboratory Techniques , Diagnostic Tests, Routine , DNA , Epidermis , Epidermophyton , Fungi , Gelatin , Hair , Magnesium Chloride , Microsporum , Polymerase Chain Reaction , Sensitivity and Specificity , Taq Polymerase , TineaABSTRACT
BACKGROUND: Trichophyton rubrum is the most common dermatophyte isolated from human and has ability to invade the tissues such as stratum comeum, nail and hair. The potential role of proteinases as virulence factors of F rMSrMm has been discussed at length. OBJECTIVE: As a first step towards assessing its virulence role, we report on the purification and characterization of proteinase from T. rubrum isolate culture filtrates. METHODS: An extracellular serine proteinase has been purified from culture filtrates of Trichophyton rubrum HP-9 by ultrafiltration, gel filtration chromatography, and affinity column chromatography. Azocoll and keratin azure were employed as the substrates of enzyme activities. Peak of proteolytic activity was analyzed by gelatin co-polymerized gel electrophoresis. RESULTS: The molecular weight of the purified enzyme was approximately exhibited to 14.0 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and molality of 14.0 kDa proteinase activity was 6.0 and 100mM, respectively. The activity was inhibited by serine proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). The proteinase degraded gelatin, collagen type VI, and keratin from human epidermis but not hemoglobin. CONCLUSION: The 14,000 Mr extracellular serine proteinase purified from T. rubrum NP-9 culture filtrates has neutral pH optimum 6.0 and activities against gelatin, collagen type VI, and keratin.