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      Characterization of melanin and optimal conditions for pigment production by an endophytic fungus, Spissiomyces endophytica SDBR-CMU319

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          Abstract

          Melanin is a natural pigment that is produced by filamentous fungi. In this study, the endophytic species, Spissiomyces endophytica (strain SDBR-CMU319), produced a brown-black pigment in the mycelia. Consequently, the pigment was extracted from the dried fungal biomass. This was followed by pigment purification, characterization and identification. Physical and chemical characteristics of the pigment showed acid precipitation, alkali solubilization, decolorization with oxidizing agents, and insolubility in most organic solvents and water. The pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and electron paramagnetic resonance spectra analyses. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen, and therefore, was not 3,4-dihydroxyphenylalanine melanin. Inhibition studies involving specific inhibitors, both tricyclazole and phthalide, and suggest that fungal melanin could be synthesized through the 1,8-dihydroxynaphthalene pathway. The optimum conditions for fungal pigment production from this species were investigated. The highest fungal pigment yield was observed in glucose yeast extract peptone medium at an initial pH value of 6.0 and at 25°C over three weeks of cultivation. This is the first report on the production and characterization of melanin obtained from the genus Spissiomyces.

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          Biosynthesis and Functions of Fungal Melanins

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            The contribution of melanin to microbial pathogenesis.

            Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of pathogenic bacteria, fungi and helminths. The study of melanin is difficult because these pigments defy complete biochemical and structural analysis. Nevertheless, the availability of new reagents in the form of monoclonal antibodies and melanin-binding peptides, combined with the application of various physical techniques, has provided insights into the process of melanization. Melanization is important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Melanin appears to contribute to virulence by reducing the susceptibility of melanized microbes to host defence mechanisms. However, the interaction of melanized microbes and the host is complex and includes immune responses to melanin-related antigens. Production of melanin has also been linked to protection against environmental insults. Interference with melanization is a potential strategy for antimicrobial drug and pesticide development. The process of melanization poses fascinating problems in cell biology and provides a type of pathogenic strategy that is common to highly diverse pathogens.
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              Pathogenic roles for fungal melanins.

              Melanins represent virulence factors for several pathogenic fungi; the number of examples is growing. Thus, albino mutants of several genera (in one case, mutated precisely in the melanizing enzyme) exhibit decreased virulence in mice. We consider the phenomenon in relation to known chemical properties of melanin, beginning with biosynthesis from ortho-hydroquinone precursors which, when oxidized enzymatically to quinones, polymerize spontaneously to melanin. It follows that melanizing intermediates are cross-linking reagents; melanization stabilizes the external cell wall against hydrolysis and is thought to determine semipermeability in the osmotic ram (the appressorium) of certain plant pathogens. Polymeric melanins undergo reversible oxidation-reduction reactions between cell wall-penetrating quinone and hydroquinone oxidation states and thus represent polymeric redox buffers; using strong oxidants, it is possible to titrate the melanin on living cells and thereby demonstrate protection conferred by melanin in several species. The amount of buffering per cell approximately neutralizes the amount of oxidant generated by a single macrophage. Moreover, the intermediate oxidation state, the semiquinone, is a very stable free radical and is thought to trap unpaired electrons. We have suggested that the oxidation state of external melanin may be regulated by external Fe(II). An independent hypothesis holds that in Cryptococcus neoformans, an important function of the melanizing enzyme (apart from melanization) is the oxidation of Fe(II) to Fe(III), thereby forestalling generation of the harmful hydroxyl radical from H(2)O(2). Thus, problems in fungal pathogenesis have led to evolving hypotheses regarding melanin functioning.
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                Author and article information

                Contributors
                Role: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: MethodologyRole: SoftwareRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – review & editing
                Role: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Writing – review & editing
                Role: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                9 September 2019
                2019
                : 14
                : 9
                : e0222187
                Affiliations
                [1 ] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
                [2 ] Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, Thailand
                [3 ] Institute for Chemical Research, Kyoto University, Kyoto, Japan
                [4 ] Graduate School of Sciences and Technology for Innovation (Agriculture), Yamaguchi University, Yamaguchi, Japan
                [5 ] Academy of Science, The Royal Society of Thailand, Bangkok, Thailand
                Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie eV Hans-Knoll-Institut, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-2653-1913
                http://orcid.org/0000-0002-6485-414X
                Article
                PONE-D-19-10160
                10.1371/journal.pone.0222187
                6733467
                31498821
                b7ccef69-3637-4ed5-b2d7-a70ec29066a3
                © 2019 Suwannarach 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.

                History
                : 9 April 2019
                : 25 August 2019
                Page count
                Figures: 5, Tables: 2, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100007858, Faculty of Science, Chiang Mai University;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100012527, Office of the Higher Education Commission;
                Award ID: BDC-PG2-159010
                Award Recipient :
                Funded by: Japan Society for the Promotion of Science
                Award Recipient :
                This work was supported by: Chiang Mai University, Thailand for Dr. Nakarin Suwannarach; Center of Excellence on Biodiversity (BDC), and The Office of The Higher Education Commission, (BDCPG2-159010) for Dr. Saisamorn Lumyong; and by Japan Society for the Promotion of Science (JSPS) in Core-to-Core Program (Establishment of an international research core for new bio-research fields with microbes from tropical area) for Dr. Kenji Matsui.
                Categories
                Research Article
                Physical Sciences
                Materials Science
                Materials
                Pigments
                Physical Sciences
                Materials Science
                Materials
                Pigments
                Organic Pigments
                Melanin
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Rice Blast Fungus
                Biology and Life Sciences
                Plant Science
                Plant Pathology
                Plant Pathogens
                Plant Fungal Pathogens
                Rice Blast Fungus
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Biology and Life Sciences
                Agriculture
                Fungiculture
                Physical Sciences
                Chemistry
                Chemical Reactions
                Chemical Precipitation
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Electron Spin Resonance Spectroscopy
                Custom metadata
                All relevant data are within the paper.

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