프로테오믹스 기법을 이용한 피부사상균 배양 여과물내 세포외단백의 분석

Abstract

Background: Proteases from dermatophytes have an important role in pathogenicity of these fungi as they facilitate penetration and colonization in the keratin structures of the stratum corneum, nails, and hair. Since the 1960s, efforts have been made to isolate and purify these enzymes, shedding light on hydrolytic properties and other characteristics of these extracellular proteins. It is now well known that under certain conditions, various proteases are produced which possess the capacity to digest casein, collagen, elastin, bovine serum albumin, hair, and keratin, while specific nature of these enzymes still remains to be elucidated. Objective: We aimed to draw a proteome map of extracellular proteins from common dermatophytes using 2-dimensional electrophoresis, thus verifying the nature and interspecies differences in composition of extracellular proteins. Methods: Following strains of dermatophytes were isolated from patients who visited the dermatologic outpatient clinic of Severance hospital and subcultured for 2 weeks on Sabouraud's dextrose agar: 2 strains of Trichophyton rubrum, 2 T. mentagrophytes, and 2 Microsporum canis. For growth media, glucose-peptone broth was added to each strain and 10 ml of media was taken, filtered using a 0.4μm syringe filter and the protein content obtained was concentrated by an ultrafiltration device before electrophoresis on the culture day 0 and 10. Stained with silver nitrate, the gel was scanned and analysed. Results: 7 spots have increased in intensity including a 16 kDa-spot with isoelectric point at 9.3 from the supernatants of M. canis culture media, 4 spots have increased including a 32 kDa-spot with isoelectric point at 6.7, from the supernatants of T. mentagrophytes, and 5 spots including a 10 kDa-spot with isoelectric point at 6.3 showed significant increase from the supernatants of T. rubrum. M. canis and T. mentagrophytes subspecies shared 2 spots that increased. Conclusion: We concluded dermatophyte fungi produce different proteins according to their subspecies, and that proteomics appears to be a useful tool for comparative analysis of dermatophyte extracellular proteins.