The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur Sirida Youngchim 1 *, Joshua D. Nosanchuk 2 , Soraya Pornsuwan 3 , Susumu Kajiwara 4 , Nongnuch Vanittanakom 1 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: syoungchim@gmail.com Introduction Malassezia species are lipophilic yeasts that are frequent components of the skin microflora of humans and most warm- blooded animals [1], [2]. Based on molecular data and lipid requirements, seven Malassezia species are now recognized [3], [4]. In humans, pityriasis versicolor (PV), known also as tinea versicolor, is a common superficial skin infection restricted to the stratum corneum that occurs when Malassezia yeast cells convert to a pathogenic mycelial form. M. globosa and M. furfur are significantly more common causes of PV than other species [5]. The disease is characterized by the presence of fine scaly patches or macules, which may be either hypo- or hyperpigmented, that are normally located on the upper parts of trunk, neck, and arms [5], [6]. PV is one of the most common pigmentary disorders worldwide, but it is more common in tropical climates, reaching as high as 50% in some tropical areas, which is attributed to the growth benefit achieved in the relative high temperature and humidity of these regions [5]. Since PV is one of the most common human skin infections, investigation into pathophysiological mechanisms underlying the disease can lead to a greater understanding of the causes, progression and outcomes of human Malassezia infections. However, to date, there is relatively little information on the virulence factors of Malassezia spp. The attributes of Malassezia yeasts implicated thus far in pathogenesis include lipolytic enzymes that injure host tissues and provide nutrients for the fungus [729], high lipid content of cell walls that protects Malassezia yeasts from phagocytosis [10] and downregulates the inflammatory immune response [11], [12], hyphae formation, and production of a tryptophan-dependent pigment that acts as protective barrier against the UVA and UVB spectrum [13], [14]. Melanins are biologically prominent macromolecules that are dark brown and black pigments formed by oxidative polymeriza- tion of phenolic compounds. Melanins can be classified into 3 typical types: eumelanin; formed by a complex polymerization process involving quinones and free radicals, phaeomelanin; derived from tyrosine and cysteine and allomelanins; formed from nitrogen free precursors [15]. Generally, two types of melanins, 1,8 dihydroxynaphthalene (DHN) and L-3,4- dihydroxyphenylalanine (DOPA) melanins are found in fungi, but most are synthesized from DHN-melanins. Melanins produced from acetate via the polyketide synthase pathway are normally black or brown and are referred to as DHN melanins. DOPA melanins are catalyzed by phenoloxidases (such as tyrosinases, laccases, or catecholases) and are called eumelanins [16]. They have been linked with virulence in an array of human pathogen fungi, such as Cryptococcus neoformans [17219], Aspergillus fumigatus [20224], Histoplasma capsulatum [25] and Penicillium marneffei [26]. Melanin has been shown to protect fungi in several PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e63764