Cryptococcus neoformans melanization incorporates multiple catecholamines to produce polytypic melanin

Abstract

Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors. Melanin is a major virulence factor in pathogenic fungi that enhances the ability of fungal cells to resist immune clearance. Cryptococcus neoformans is an important human pathogenic fungus that synthesizes melanin from exogenous tissue catecholamine precursors during infection, but the type of melanin made in cryptococcal meningoencephalitis is unknown. We analyzed the efficacy of various catecholamines found in brain tissue in supporting melanization using animal brain tissue and synthetic catecholamine mixtures reflecting brain tissue proportions. Solid-state NMR spectra of the melanin pigment produced from such mixtures yielded more melanin than expected if only the preferred constituent dopamine had been incorporated, suggesting uptake of additional catecholamines. Probing the biosynthesis of melanin using radiolabeled catecholamines revealed that C. neoformans melanization simultaneously incorporated more than one catecholamine, implying that the pigment was polytypic in nature. Nonetheless, melanin derived from individual or mixed catecholamines had comparable ability to protect C. neoformans against ultraviolet light and oxidants. Our results indicate that melanin produced during infection differs depending on the catecholamine composition of tissue and that melanin pigment synthesized in vivo is likely to accrue from the polymerization of a mixture of precursors. From a practical standpoint, our results strongly suggest that using dopamine as a polymerization precursor is capable of producing melanin pigment comparable to that produced during infection. On a more fundamental level, our findings uncover additional structural complexity for natural cryptococcal melanin by demonstrating that pigment produced during human infection is likely to be composed of polymerized moieties derived from chemically different precursors. Cryptococcus neoformans is an encapsulated, free-living fungal organism that can establish opportunistic infections in both plant and animal hosts (1Springer D.J. Mohan R. Heitman J. Plants promote mating and dispersal of the human pathogenic fungus Cryptococcus.PLoS One. 2017; 12e0171695Crossref PubMed Scopus (20) Google Scholar). C. neoformans is broadly prevalent in the environment and colonizes a variety of ecological niches, which notably include soil and bird guano (2Lin X. Heitman J. The biology of the Cryptococcus neoformans species complex.Annu. Rev. Microbiol. 2006; 60: 69-105Crossref PubMed Scopus (280) Google Scholar). Despite ubiquitous exposure of the human population, C. neoformans rarely causes disease in individuals with intact immunity as the infection is either cleared or becomes asymptomatically latent (3Coelho C. Bocca A.L. Casadevall A. The intracellular life of Cryptococcus neoformans.Annu. Rev. 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Casadevall A. Microbiol. 6: PubMed Scopus Google the of this in to melanin produced in the and of melanin produced during the of C. neoformans infection. The of cryptococcal disease results in brain which a and of the for of precursors. Although melanin can be from human and animal tissue J.D. A.L. Casadevall A. of Cryptococcus neoformans in human brain Full Text Full Text PDF PubMed Scopus Google Scholar, A.L. J.D. M. Casadevall A. of polymerized melanin by Cryptococcus neoformans in Immun. PubMed Scopus Google Scholar), the of that can be from animal of infection this to develop a system that the of catecholamine precursors in the human on that catecholamines from human in the of and in human cerebral using and PubMed Scopus Google Scholar, of in human for in as as in Res. PubMed Scopus Google Scholar), C. neoformans in a mixture of catecholamines to brain of and the and of melanin produced from this brain catecholamine mixture and that C. neoformans can precursors simultaneously during melanin is found to be comparable to melanin in ability to protect C. neoformans from of such as and Despite the ability of C. neoformans to a mixture of chemically precursors during melanin for this depending on the of precursors and structural We to the melanization during infection by C. neoformans in a mixture of catecholamines of human brain in this was the of a that be capable of melanin from the human of derived from the of C. neoformans and virulence in and melanization on as a result of N. F. of the and of Cryptococcus neoformans and to virulence 2014; PubMed Scopus Google Scholar). and that derived from of through the central nervous system are more and more melanin on N. 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We melanization by and on with as as the individual precursors and and The in the primary for was as a C. neoformans and been to with of the cryptococcal PubMed Scopus Google Scholar, of and in Cryptococcus PubMed Scopus Google Scholar). a of melanin and a of for both and with of which precursors melanization was with that of of for the after for left However, cells than after and only melanization after an additional of by on was comparable to the with of the individual catecholamines left and was more than the melanin of to in the of the human of the of C. neoformans was for the melanin produced by C. neoformans with a mixture of catecholamine was in for which is the of in with or either or more for cells in with but both melanization after The in more but an comparable to the after was for in with or the human of pigment more and a comparable for melanin for cells from and to and and in was by light was to be and to as melanin to be on yielded revealed in the of derived from The melanin from the and in to those from in J. J.D. O. T. Casadevall A. of Cryptococcus neoformans from cells in different PubMed Scopus Google Scholar). intact from the and to be and was an of with that of The melanin from the the both in with a mixture of intact and more and in of the of of melanin using revealed with of or that in the Melanin from and in by for and for and with those The is with of of and in Cryptococcus PubMed Scopus Google Scholar). An of intact in of the of was from cells on brain tissue light of this the pigment in our especially for melanin that that produced in human brain tissue but in to structural the of the derived from C. neoformans in to melanin to the of this Solid-state NMR was to and with that of melanin from cells with either of the major constituent or The spectra of of of the that are of melanin S. R. S. Casadevall A. NMR to the of Cryptococcus neoformans melanization with different catecholamine PubMed Scopus Google Scholar, S. R. Casadevall A. 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The of melanin which in C. neoformans are deposited and the this from the during melanin this melanin which of the in to than only the fungal Melanin spectra that to in to those that to the melanin pigment The that in spectra that the from C. neoformans with are melanin Our C. neoformans is able to more than one melanization precursor the pigment with The of the melanin which results in the of that are to a melanin synthesized from or from was that the and melanin but the of the of to and the to the pigment to the in the melanin in to the both from that of the precursors. was had that precursors such as or result in more melanin in to such as or S. R. S. Casadevall A. NMR to the of Cryptococcus neoformans melanization with different catecholamine PubMed Scopus Google Scholar, S. R. Casadevall A. Solid-state NMR the of Cryptococcus neoformans fungal in the Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. S. C. A. Casadevall A. The melanization more on melanin in and fungal Full Text Full Text PDF PubMed Scopus Google Scholar). in results in melanin that a pigment to the such as and The of the melanin which from a and pigment with to that on of only the suggesting of the additional catecholamines. spectra the of pigment in the melanin this was in both the and spectra of melanin from C. neoformans cells the in melanin that this of the pigment synthesized from different our that C. neoformans can different melanization precursors are our structural for the of our by radiolabeled catecholamines as a and of both and can be incorporated the melanin pigment the C. neoformans in the of a of with and the of or in the primary was with the of as a the of in the was more in the from the additional precursors with that of the The of a of either or in a in and a in the of We to the of melanization for and resulting from of the Casadevall A. The of on Cryptococcus neoformans and PubMed Scopus Google Scholar). a comparable in was with of that C. neoformans can more than one type of precursor that the with C. neoformans from the of the radiolabeled precursors cells that had been in mixtures of radiolabeled and precursors in to melanin to of the was in melanin from cells with either or The of of either precursor melanin was different melanization was by an of a catecholamine or a mixture of and in either a of the was after of C. neoformans in was to from the of Casadevall A. of Cryptococcus neoformans to Microbiol. 60: PubMed Scopus Google Scholar). the of the brain mixture of cells for in or and with cells for after of with melanization in was than with the precursors in from or in cells from of and melanin is a virulence factor that to C. neoformans human to the ability of melanin to against An in system that I. J. and virulence of Cryptococcus Immun. 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Casadevall A. of Cryptococcus neoformans in human brain Full Text Full Text PDF PubMed Scopus Google Scholar, A.L. J.D. M. Casadevall A. of polymerized melanin by Cryptococcus neoformans in Immun. PubMed Scopus Google Scholar). We from cells on with a brain catecholamine but only a of the cells was as melanin such as dopamine in brain tissue are and by an found in cells J. K. of dopamine PubMed Scopus Google Scholar). to the using brain tissue as the of the of catecholamines in brain tissue with the in of the precursors in and of catecholamines during the cells are light of for C. neoformans in a mixture of catecholamines to in the human brain and melanization on both and in We more melanization by the with especially the human of and this is with the melanization and virulence N. F. of the and of Cryptococcus neoformans and to virulence 2014; PubMed Scopus Google Scholar). 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