The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

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The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur Sirida Youngchim1*, Joshua D. Nosanchuk2, Soraya Pornsuwan3, Susumu Kajiwara4, Nongnuch Vanittanakom1 1 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 2 Departments of Medicine (Infectious Diseases) and Microbiology/ Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Physical Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand, 4 Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan Abstract Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization. Citation: Youngchim S, Nosanchuk JD, Pornsuwan S, Kajiwara S, Vanittanakom N (2013) The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur. PLoS ONE 8(6): e63764. doi:10.1371/journal.pone.0063764 Editor: Floyd L. Wormley, The University of Texas at San Antonio, United States of America Received December 27, 2012; Accepted April 5, 2013; Published June 7, 2013 Copyright: ß 2013 Youngchim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Research Fund of Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand. JDN is partially supported by NIH AI52733. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction yeasts implicated thus far in pathogenesis include lipolytic enzymes that injure host tissues and provide nutrients for the fungus [729], Malassezia species are lipophilic yeasts that are frequent high lipid content of cell walls that protects Malassezia yeasts from components of the skin microflora of humans and most warm- phagocytosis [10] and downregulates the inflammatory immune blooded animals [1], [2]. Based on molecular data and lipid response [11], [12], hyphae formation, and production of a requirements, seven Malassezia species are now recognized [3], [4]. tryptophan-dependent pigment that acts as protective barrier In humans, pityriasis versicolor (PV), known also as tinea against the UVA and UVB spectrum [13], [14]. versicolor, is a common superficial skin infection restricted to the Melanins are biologically prominent macromolecules that are stratum corneum that occurs when Malassezia yeast cells convert to dark brown and black pigments formed by oxidative polymeriza- a pathogenic mycelial form. M. globosa and M. furfur are tion of phenolic compounds. Melanins can be classified into 3 significantly more common causes of PV than other species [5]. typical types: eumelanin; formed by a complex polymerization The disease is characterized by the presence of fine scaly patches process involving quinones and free radicals, phaeomelanin; or macules, which may be either hypo- or hyperpigmented, that derived from tyrosine and cysteine and allomelanins; formed from are normally located on the upper parts of trunk, neck, and arms nitrogen free precursors [15]. Generally, two types of melanins, 1,8 [5], [6]. PV is one of the most common pigmentary disorders dihydroxynaphthalene (DHN) and L-3,4- dihydroxyphenylalanine worldwide, but it is more common in tropical climates, reaching as (DOPA) melanins are found in fungi, but most are synthesized high as 50% in some tropical areas, which is attributed to the from DHN-melanins. Melanins produced from acetate via the growth benefit achieved in the relative high temperature and polyketide synthase pathway are normally black or brown and are humidity of these regions [5]. referred to as DHN melanins. DOPA melanins are catalyzed by Since PV is one of the most common human skin infections, phenoloxidases (such as tyrosinases, laccases, or catecholases) and investigation into pathophysiological mechanisms underlying the are called eumelanins [16]. disease can lead to a greater understanding of the causes, They have been linked with virulence in an array of human progression and outcomes of human Malassezia infections. pathogen fungi, such as Cryptococcus neoformans [17219], Aspergillus However, to date, there is relatively little information on the fumigatus [20224], Histoplasma capsulatum [25] and Penicillium virulence factors of Malassezia spp. The attributes of Malassezia marneffei [26]. Melanin has been shown to protect fungi in several PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e63764 Melanin and Mycelial Production in M. furfur ways. It functions as a beneficial physiologic redox buffer, protects plasts. The protoplasts were collected by centrifugation, washed pathogens against environmental stress such as UV radiation, three times with PBS, and incubated in 4.0 M guanidine temperature extremes, hydrolytic enzymes, metals or free radicals, thiocyanate (Sigma) overnight at room temperature. The resultant and enhances resistance to antifungal drugs [19]. dark particles were collected by centrifugation, washed three times The study of melanization in Malassezia spp. has previously been with PBS, and treated with 1.0 mg/ml Proteinase K (Roche) in investigated using the Masson-Fontana stain, which demonstrated reaction buffer [10.0 mM Tris, 1.0 mM CaCl2 and 0.5% (w/v) the accumulation of black pigment on the cell wall of yeast cells SDS, pH 7.8] and incubated at 37uC. The resultant debris was both in vitro and during infection [27]. However, the Masson– washed three times with PBS and boiled in 6.0 M HCl for 1.5 Fontana silver stain is not specific for melanins, as evidenced by hours. After treatment by boiling in acid, melanin particles were the fact that both melanized and non-pigmented C. neoformans cells collected by filtration through Whatman paper no. 1 and were are stained by this method [28]. In this report, we have confirmed washed extensively with distilled water. Melanin particles were the presence of melanins in vitro-melanized yeast cells of M. furfur then dialyzed against distilled water for 10 days until the acid was by utilizing techniques developed to study and isolate melanins completely removed, whereupon they were lyophilized. from other fungal pathogens. In addition, a melanin-specific monoclonal antibody (MAb) is used in the confirmation of Electron Spin Resonance (ESR) Spectroscopy Analyses melanization both in vitro and during human infection. We show ESR spectroscopy is a technique that directly detects the signal that M. furfur produces DOPA-melanin. We also show that kojic from a free electron spin or free radical. Due to the abundance of acid, a tyrosinase inhibitor, can trigger the mycelial transformation stable free radicals in melanin pigments, ESR is used as a tool to in M. furfur, which is considered to be important in the indicate the presence of melanin in the samples by using a Gunn pathogenesis of Malassezia. diode as a microwave source [32]. The ESR signal of melanin in derivative mode shows a typical single peak located in the middle Materials and Methods of the spectrum at approximately 3500 Gauss. We used this Fungal Strain and Media method with a total of 2 g freeze-dried material in each case M. furfur NBRC 0656 was maintained by monthly subculture on (analysis was carried out in silica cuvettes). Aspergillus fumigatus and Modified Dixon medium (mDixon; 1 liter of distilled water, 36 gm P. marneffei melanins were used as positive controls [24], [26]. malt extract, 6 gm peptone, 20 gm ox bile (all obtained from Difco), 10 ml Tween 40, 2 ml glycerol, 2 ml olive oil (all obtained Immunofluorescence Analyses of Melanin in M. furfur from Sigma) 0.05 gm chloramphenicol (Amresco), and 15.0 gm in vitro agar (Difco) with a pH 6.0 (modified from Guillot and colleague Suspensions of yeast cells from M. furfur grown on mDixon or [29]). Unless otherwise specified, M. furfur was cultured on MM with L-DOPA were air-dried on poly-L-lysine slides, and chemically defined minimal medium (MM) agar (15.0 mM then incubated with Superblock Blocking Buffer in PBS (Pierce) glucose, 10.0 mM MgSO4, 29.4 mM KH2PO4, 13.0 mM glycine, for 2 h at 37uC or overnight at 4uC to block non-specific binding. 10.0 mM NH4Cl and 3.0 mM thiamine; pH 5.5) and supplement- The slides were washed three times with PBS, then incubated for ed with 0.1% (v/v) Tween 40, 0.1% (v/v) Tween 80 and 1.5% 1.5 h at 37uC with 10 mg/ml of anti-melanin MAb 8D6 (against agar. For melanization studies, M. furfur was incubated at 30uC for melanin of A.
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