Pathogenic roles for fungal melanins

Abstract

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.

FIG. 1

Structure of squid melanin proposed by Nicolaus et al. (47). Rings are aromatic. Each pair of o-oxygen substutituents can exist as either quinone, hydroquinone, or semiquinone groups.

FIG. 2

Electron micrograph of a “melanin ghost” of C. neoformans. Organs from a mouse infected with Mel⁺ C. neoformans were extracted with solvents, digested in hot HCl, and centrifuged. These structures were not seen when a Mel⁻ infection was so treated. Courtesy of Rosas et al. (For a related study, see reference 71).

FIG. 3

Oxidation states of the quinonic residues of melanin: the hydroquinone, the free radical semiquinone, and the quinone.

FIG. 4

Redox buffering by melanin. The electrochemical potential of an inert electrode was rhythmically cycled between positive and negative (indicated on horizontal axis) in the presence of the dissolved melanin precursor, 5,6-dihydroxyindole. The precursor was incrementally oxidized and melanin was incrementally precipitated with each positive sweep. Current to and from the electrode was recorded on the vertical axis. Because voltage varied linearly with time, the horizontal “voltage axis” is also a time axis. Since charge equals current times time and is proportional to area, the increasing area circumscribed indicates that buffer capacity increases with melanin film thickness (22). Reprinted from reference .

FIG. 5

Permanganate titration of washed cells of C. neoformans. Absorbance at 520 nm (O.D. 520) was read after 3 h of incubation of cells with the oxidant. Squares indicate permanganate in buffer; diamonds indicate nonmelanized cells; circles indicate melanized cells. The minimal fungicidal concentrations (arrows) corresponded to the equivalence points of the redox titrations.