Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of pathogenic bacteria, fungi and helminthes. 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.
The ability of certain microbes to produce melanin has been linked with virulence and pathogenicity for their respective animal or plant hosts. Microbes capable of synthesizing melanin pigments include diverse fungal, bacterial and helminthic pathogens. The mechanism by which melanin contributes to virulence has been most extensively studied for fungal pathogens and, consequently, the experience with fungal pathogens is prominently discussed. As several comprehensive reviews of melanogenesis in fungi and the contribution of melanin to fungal virulence have been published recently (Butler and Day, 1998; Henson et al., 1999; Casadevall et al., 2000; Jacobson, 2000; Hamilton and Gomez, 2002), we have chosen to review the broad subject of microbial melanization and virulence with emphasis on highlighting common mechanisms and establishing links between otherwise unrelated observations. The contribution of melanin to microbial pathogenesis is viewed from the vantage point of its contribution to the outcome of the host–pathogen interaction.
What are melanins?
A discussion of the contribution of melanins to microbial pathogenesis must begin with the definition of melanin. It is noteworthy that despite their great abundance in the terrestrial biomass, remarkably little is known about the structure of melanins. A substance is considered to be a melanin if it is dark in colour, insoluble in aqueous or organic fluids, resistant to concentrated acid and susceptible to bleaching by oxidizing agents (Nicholaus et al., 1964; Prota, 1992; Butler and Day, 1998). Melanins are believed to be composed of polymerized phenolic and/or indolic compounds (Henson et al., 1999). Unfortunately, a more rigorous definition of melanins is not possible at this time because these pigments remain poorly characterized. The problem with defining melanins is that current biochemical and biophysical techniques are unable to provide a chemical structure for this complex polymer because they are amorphous, insoluble and not amenable to solution or crystallographic structural studies. No methods have been established that permit the reliable identification of the monomer units that comprise the polymer. Consequently, the full understanding of the structural and biochemical properties of melanins is beyond our current analytical horizon. Nevertheless, a variety of analytical techniques have been used to study melanins, which have provided important information on its structure and properties. Melanins are negatively charged, hydrophobic pigments of high molecular weight (White, 1958; Nosanchuk and Casadevall, 1997; Nosanchuk et al., 1999b; Jacobson, 2000). An operational definition for a pigment as a melanin can be provided by electron spin resonance (ESR) characteristics which include establishing that a compound is a stable free radical (Enochs et al., 1993). Methods for partial chemical degradation of melanin followed by HPLC microanalysis have been developed and are useful in the characterization of specific types of melanin (Wakamatsu and Ito, 2002; Wakamatsu et al., 2002).
Melanin synthesis in mammals is catalysed by tyrosinase (Sanchez-Ferrer et al., 1995; del Marmol and Beermann, 1996). The mammalian pathway can utilize tyrosine or dihydroxyphenylalanine to synthesize melanin. Microbes can synthesize melanin via phenoloxidases (such as tyrosinases, laccases or catacholases) and/or the polyketide synthase pathway. In contrast to tyrosinases, laccases are unable to convert tyrosine to 3,4-dihyroxyphenylalanine (L-DOPA) (Wheeler and Bell, 1988). Laccases may vary in their capacity to catalyse the polymerization of diverse phenolic substrates to melanin (Kwon-Chung et al., 1983). Melanins are generally black or brown pigments, although other colours may occur (Hill, 1992). Melanins derived from L-DOPA are referred to as eumelanins and are characteristically black or brown. Yellow or reddish melanins are called pheomelanins and incorporate cysteine with L-DOPA. Brownish melanins formed from homogentisic acid by tyrosinases are called pyomelanins (Yabuuchi and Ohyama, 1972). Pigments derived from acetate via the polyketide synthase pathway are generally black or brown and referred to as dihydroxynaphthalene (DHN) melanins
Functions of melanin in biological systems
Melanins are ubiquitous in the biological kingdoms (Hill, 1992) and many of the dark pigments in nature are considered melanins (Wheeler and Bell, 1988). A great variety of functions have been ascribed to melanins. Melanins can serve as energy transducers, bind diverse drugs and chemicals and affect cellular integrity (reviewed in Hill, 1992). Melanin is also used for camouflage and sexual display. The ink of the cuttlefish Sepia officinalis is a suspension of melanin particles. The coloration in black and red hair arises from different types of melanin (Castanet and Ortonne, 1997) and melanins are responsible for feather coloration in certain birds (McGraw and Hill, 2000). Melanins in melanocytes in skin provide protection against sunlight and almost certainly contribute to the resistance of melanoma to therapeutic radiation (Hill, 1991). Melanins have in vitro activity against human immunodeficiency virus (Montefiori and Zhou, 1991) and can inhibit syncytium formation and cytopathic effects of the virus (Sidibe et al., 1996). Insects use melanin for host defence in a mechanism whereby the polymer is produced to wall off microbial intruders (Richman and Kafatos, 1996). In this system, tissue damage by microbial pathogens activates a prophenoloxidse in the haemolymph to encase the bacterial, protozoal or fungal pathogens in melanin (Marmaras et al., 1996). The role of melanin continues to be enigmatic in other circumstances. For example, the function of melanin in neurons of the substantia nigra in the human brain is unknown (Zecca et al., 2001; 2002).
Melanization, and its consequences on mammalian virulence, has been most extensively studied in C. neoformans and this fungus will serve as a paradigm for this review. C. neoformans is a yeast-like fungus that is commonly found in soils contaminated with bird excreta. Cryptococcal infection is thought to be common and to be acquired in childhood, but disease (e.g. cryptococcosis) is relatively rare (Goldman et al., 2001). The most common presentations of cryptococcal disease are pneumonia and meningo-encephalitis. The prevalence in the United States of cryptococcal meningo-encephalitis in individuals with AIDS is estimated to be 5%-10% (Currie and Casadevall, 1994; Mitchell and Perfect, 1995) and is significantly higher in underdeveloped countries. However, immune reconstitution with highly active retroviral therapy appears to reduce the incidence of cryptococcosis. The ability of C. neoformans to make pigments was discovered by F. Staib in the early 1960s when he noted that cryptococcal colonies in bird seed (Guizotia abyssinica) agar turned brown (Staib, 1962; 1963). When grown in liquid culture with the addition of phenolic compounds, melanin comprises approximately 15% of the dry weight of C. neoformans (Wang and Casadevall, 1996a).
The production of melanin in C. neoformans requires the presence of exogenous substrate in the form of certain o-diphenolic or p-diphenolic compounds, such as L-DOPA (Kwon-Chung et al., 1983). Expression of laccase is induced in response to glucose starvation (Nurudeen and Ahearn, 1979; Polacheck et al., 1982), vesicular acidification (Erickson et al., 2001) and temperatures less than 37°C (Jacobson and Emery, 1991a). Pigmentation in other fungi, such as Ustilago hordei, is also affected by the availability of nutrients, pH and temperature (Lichter and Mills, 1998). In C. neoformans, oxidation of o-diphenols with hydroxyl groups in the 2,3- or 3,4-positions results in the deposition of insoluble melanin in the cell wall whereas oxidation of p-diphenols with hydroxyl groups in the 1,4- or 2,5-positions results in the production of soluble pigments that diffuse into the medium (Chaskes and Tyndall, 1975). The pigment produced by C. neoformans was shown to be a melanin by ESR criteria (Wang et al., 1995; Wang and Casadevall, 1996a) and by chemical characterization of pigment degradation products (Williamson et al., 1998). The neurotropism of C. neoformans may be associated with its ability to utilize catacholamines for melanin synthesis (Polacheck et al., 1982). Dopamine, adrenaline and noradrenaline are each found in the central nervous system where they function as neurotransmitters and areas rich in these molecules, such as the basil ganglia, are often involved in infection with C. neoformans (Lee et al., 1996).
Although the structure of C. neoformans melanin remains elusive, some characteristics of the eumelanin have been defined. Chemical composition studies of melanin isolated from C. neoformans show that the C : N : O ratio is very similar to that of L-DOPA, consistent with the view that the synthesis of this pigment results from polymerization of L-DOPA derivatives (Wang and Casadevall, 1996a; Nosanchuk and Casadevall, 1997; Rosas et al., 2000a). Zeta potential studies have documented that the polymer is highly negatively charged (Nosanchuk and Casadevall, 1997). Analysis of the amino acids of peptides that bind C. neoformans melanin revealed a motif rich in positively charged and aromatic residues (Nosanchuk et al., 1999b). The presence of positively charged amino acids in melanin-binding peptides is consistent with what would be expected in a protein structure that binds to a negatively charged surface. The predominance of aromatic residues in the peptide, combined with the fact that melanin originates from L-DOPA, suggests the possibility of aromatic–aromatic interactions during peptide binding to melanin.
Laccases may also promote virulence by mechanisms other than catalysing the synthesis of melanin. It was proposed that the laccase of C. neoformans promotes virulence by inhibiting the oxidative burst in the phagosomal space of macrophages as a consequence of reducing Fe3+ to Fe2+ (Liu et al., 1999a). Cryptococus neoformans laccase was recently been shown to be tightly associated with the cell wall by a hydrolysable bond (Zhu et al., 2001). Data from Pleurotus ostreatus suggest that extracellular proteases may affect concentrations of secreted laccases cleaved from the cell wall (Palmieri et al., 2001). The putative location of laccase in C. neoformans is consistent with its interference with hydroxyl radical production by macrophages (Liu et al., 1999a) and may also permit oxidation of exogenous catecholamines (Zhu et al., 2001) that are potentially toxic to the organism (Tse et al., 1976).
Studies in other microbes indicate additional functions of laccase. Because of their ability to oxidize diverse compounds such as Mn2+ to Mn3+ (Brouwers et al., 1999; Hofer and Schlosser, 1999) and humic acids (Chefetz et al., 1998; Scheel et al., 1999), fungal laccases are widely used in biotechnology in applications such as transformation of antibiotics and steroids, textile dye bleaching, pulp delignification and removal of phenolics from wine (Luisa et al., 1996). Laccase is essential for the lignolytic ability of Pycnoporus cinnabarinus (Eggert et al., 1996a; b; Eggert et al., 1997; Temp et al., 1999). Pycnoporus cinnabarinus laccase also catalyses the formation of cinnabarinic acid, a compound with antibacterial activity that may protect the fungus against environmental microbial predators (Eggert et al., 1995; Eggert, 1997). Laccases have been extensively studied for use in remediation of xenobiotic effluents (D’2Annibale et al., 1998; Kissi et al., 2001). The capacity of laccase to serve in bioremediation is exemplified by the ability of the white rot fungus Pleurotus ostreatus together with the mediator 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) (ABTS) to completely and rapidly degrade the nerve agents VX and Russian VX (RVX) (Amitai et al., 1998).
Polyketide pathway and 1,8-dihydroxynaphthalene (DHN) melanin
Most fungi in soil are melanized (Hunt and Fogel, 1983) and the production of this pigment may protect the organisms from diverse environmental insults. The importance of fungal melanization as a metabolic product is suggested by the fact that melanotic fungi often produce large quantities of melanin. For example, melanin accounts for 30% of the dry weight of Agaricus bisporus (Rast and Hollenstein, 1977). The microbiota at the site of the Chernobyl nuclear reactor accident provides one of the most striking associations between fungal melanogenesis and the ability of these organisms to survive in an extreme environment (Mironenko et al., 2000). Since the accident, the prevalence of black fungi in the contaminated soil surrounding Chernobyl has dramatically increased (Zhdanova et al., 1991; 1994). Remarkably, over 37 species of fungi have been identified within the damaged reactor, including A. alternata and Cladosporium sphaerospermum, within this site where radiation levels are over 10 thousand times the lethal human dose (Zhdanova et al., 2000). Genetic analysis of A. alternata strains inhabiting the reactor revealed significant homology, whereas environmental strains from outside the contaminated region were genetically diverse. (Mironenko et al., 2000). This finding is consistent the extreme conditions present in the Chernobyl environment selecting for a radiation tolerant A. alternata clone (Mironenko et al., 2000).
Recently, interest has intensified in the study of bacterial laccases for potential biotechnological exploitation of their catalytic properties to degrade a variety of compounds, ranging from simple phenols and anilines (Xu, 1996) to polycyclic aromatic hydrocarbons and organophosphorus insecticides (Amitai et al., 1998). Bacterial laccases are attractive economically because purification of enzymes from bacteria is relatively simple and inexpensive. Although tyrosinases occur more frequently than laccases in the bacterial kingdom (Fernandez et al., 1999), laccases are still widespread in bacteria. A recent paper reported the identification of putative bacterial laccases in 14 diverse species by blast searching protein sequence databases available at http:www.ncbi.nlm.nih.govindex.html and http:www.tigr.org using fungal laccase sequences as queries (Alexandre and Zhulin, 2000). The bacteria identified included several important human pathogens, such as Escherichia coli, Pseudomonas spp., Yersinia pestis, Campylobacter jejuni, Bordetella pertussis and Mycobacterium spp. (Alexandre and Zhulin, 2000). Although not identified by that blast search, Mycobacterium leprae heretofore has been deemed unique among mycobacteria in its ability to oxidize a variety of diphenols into quinones (Prabhakaran, 1971;Prabhakaran and Harris, 1985) and L-DOPA oxidation has been used to facilitate the rapid laboratory identification of this organism (Prabhakaran et al., 1977).
The brown pigment produced by Pseudomonas aeruginosa, described as early as 1897, is a pyomelanin (Yabuuchi and Ohyama, 1972; Ogunnariwo and Hamilton-Miller, 1975). The primary structure of the tyrosinase responsible for melanin synthesis in P. aeruginosa, 4-dihydroxyphenylpyruvate dehydrogenase, has been characterized (Ruetschi et al., 1992). Melanization of P. aeruginosa may be of clinical relevance. In a study of 712 P. aeruginosa clinical isolates, 16 isolates were melanogenic (Elston, 1968). Unfortunately, no information was given in that study regarding whether melanized strains produced more severe illness. Melanogenic P. aeruginosa have a higher lecithinase and neuraminidase activity which may affect their pathogenicity (Tydel’skaia et al., 1981). In addition to 4-dihydroxyphenylpyruvate dehydrogenase, P. aeruginosa and the related species Pseudomonas putida appear to have laccases (Alexandre and Zhulin, 2000). In the related human pathogenic species, Stenotrophomonas maltophilia, melanin is constitutively synthesized intracellularly by tyrosinase (Wang et al., 2000; Ruan et al., 2002). Melanization by these pathogenic organisms merits further study.
Azospirillum lipoferum, a bacterium associated with the roots of grasses, acquires laccase activity during growth at extremely low oxygen concentrations (Alexandre and Bally, 1999). The emergence of laccase activity occurs via a two-step phenotypic switching process (Alexandre and Bally, 1999). It is possible that intermediate quinones produced during melanin formation or that melanin may provide a means for A. lipoferum to respire in an anaerobic setting (Newman and Kolter, 2000). Azospirillum brasilense produces a brown melanin-like pigment during encystment (Sadasivan and Neyra, 1987). Azospirillum chroococcum synthesizes melanin from catechols (Shivprasad and Page, 1989).
Melanin synthesis in helminths
Like the fungi and bacteria, laccase and tyrosinase activities have been described for several helminths. Fasciola gigantica has a tyrosinase type of enzyme that exists in both membrane bound and soluble forms and can oxidize mono- and diphenol compounds (Nellaiappan et al., 1989). Unlike the fungi and bacteria there has been relatively little work done on the association of melanin synthesis by protozoa and helminths and virulence for animal hosts. However, for several helminths there is indirect evidence for a role of melanin in pathogenesis as polyphenol acitivity is important in egg shell formation where melanization is presumably part of the shell hardening as the egg matures. In Schistosoma mansoni, phenoloxidase is associated with cross-linking of precursor proteins in eggshell synthesis (Johnson et al., 1987; Wells and Cordingley, 1991; Eshete and LoVerde, 1993; Ribeiro-Paes and Rodrigues, 1995). Similarly, Isoparorchis hypselobagri has a polyphenol oxidase activity associated with egg-shell formation (Srivastava and Gupta, 1978). In Trichuris suis, phenoloxidase acitivity is located on proximal eggs where it is believed to function in egg shell hardening (Fetterer and Hill, 1993; 1994). For Trichuris muris inhibition of phenol oxidase activity by disulfiram results in malformed eggs that are unable to produce murine infection (Hill and Fetterer, 1997).
Location of melanin in cells
Microbial melanin can be found in intracellular and/or extracellular (e.g. outside cell membrane) spaces. In C. neoformans, transmission electron micrographs reveal an electron dense layer in the cell wall external to the cell membrane that is absent in non-melanized cells (Wang et al., 1995). Melanized particles (‘ghosts’) isolated from in vitro pigmented cells by serial treatment with enzymes, denaturant and hot acid are similar in dimension to their parental melanized cells, consistent with their origin from cell wall polymerized melanin (Fig. 1) (Wang et al., 1995; Wang and Casadevall, 1996a). Furthermore, melanin-binding antibody and phage both labelled melanized C. neoformans cells at the cell wall (Nosanchuk et al., 1998; 1999b; 2000; Rosas et al., 2000b). Similarly, a phage display antibody to pheomelanin demonstrated that melanin was located in the septa and outer walls of Alternaria alternata conidia (Carzaniga et al., 2002). Exophiala dermatitidis cell walls are dark whereas albino mutants appear hyaline in electron micrographs (Geis et al., 1984; Wheeler and Bell, 1988) and the dark colour of the cells is lost upon removal of the cell wall (Dixon et al., 1991). In Colletotrichum lagenarium (Kubo and Furusawa, 1986; Takano et al., 1997) and Verticillium dahliae (Wheeler et al., 1976) melanin is found in layers within the cell wall. As with V. dahliae (Butler and Day, 1998) and Sporothrix schenckii (Romero-Martinez et al., 2000), melanin can also be deposited as granules at the surface of the cell wall. The finding of melanin at the cell wall is in contrast to mammalian melanocytes where melanin is located exclusively within specialized vacuoles, melanosomes (Marks and Seabra, 2001). However, in Fonsecaea pedrosoi melanin accumulates on cytoplasmic bodies comparable to melanosomes and on the outer layer of the cell wall (Alviano et al., 1991). Fungi may also produce extracellular melanins (reviewed in Wheeler and Bell, 1988).
Association of melanin with fungal virulence for mammals
Melanin synthesis is associated with virulence for mammals in several pathogenic fungi including C. neoformans (Salas et al., 1996) and E. dermatitidis (Dixon et al., 1989) by comparing the relative pathogenicity of wild-type strains to mutants incapable of melanization. For C. neoformans an extensive body of evidence from several laboratories has established a role for melanization in virulence. Melanin-deficient mutant strains generated by UV-irradiation were avirulent in murine models of cryptococcal infection (Kwon-Chung et al., 1982; Rhodes et al., 1982). Reversion to virulence was associated with recovery of melanin production (Kwon-Chung and Rhodes, 1986). Microevolution within a laboratory strain of C. neoformans resulted in the generation of isolates with attenuated virulence that had reduced melanin production as well as small polysaccharide capsules and slower growth rates (Franzot et al., 1998). Infection with a heavily pigmented strain of C. neoformans inhibited the afferent phase of the immune response as demonstrated by decreased TNF-α production and reduced lymphoproliferation compared to a weakly melanizing strain (Huffnagle et al., 1995). Intracerebral infection of mice with an albino strain of C. neoformans resulted in minimal tissue damage and induced IL-12, IL-1β and TNF-α whereas a revertant melanotic strain caused extensive damage and inhibited the cytokine response (Barluzzi et al., 2000).
The enzyme responsible for the phenoloxidase activity of C. neoformans has been purified, characterized as a typical laccase, and cloned (Ikeda et al., 1993; Williamson, 1994). The gene encoding the laccase (CNLAC1) was shown to be important in virulence by survival studies, which established that gene deletion in a wild-type strain decreased virulence and the complementation of a melanin-deficient strain increased virulence (Salas et al., 1996). The genetic regulation of melanin production is complex (Blackstock et al., 1999) and several pathways necessary for the production of the polymer have recently been identified. cAMP-associated signalling pathways have been associated with several virulence factors, including melanin (Alspaugh et al., 1997; 1998). Disruption of a gene encoding a cAMP-dependent protein kinase results in mutants that do not produce melanin or a polysaccharide capsule and are avirulent (D’Souza et al., 2001). Downregulation of the gene responsible for an inositol-phosphoryl ceramide synthase significantly impairs melanin synthesis and pathogenicity of C. neoformans (Luberto et al., 2001). Disruption of the gene encoding a vesicular proton pump results in mutant strains that have reduced melanin synthesis, capsule production, urease expression, and growth at 37°C and the mutants are avirulent in mice (Erickson et al., 2001).
Inhibition of melanin by glyphosate, a systemic herbicide, significantly prolonged survival in experimental cryptococcosis (Nosanchuk et al., 2001). Studies of cryptococcal cells isolated from infected animals treated with glyphosate demonstrated that cellular melanin was either absent or disorganized (Nosanchuk et al., 2001). Therapy with monoclonal antibodies (mAb) to melanin also significantly improved survival in lethally infected mice (Rosas et al., 2001). Mice treated with the melanin-binding mAbs had approximately 100-fold lower fungal burden than mice given an irrelevant mAb (Rosas et al., 2001). In vitro studies demonstrated that melanin-binding mAbs completely abrogated cell growth, whereas there was no effect on the replication of non-melanized cells (Rosas et al., 2001). This affect appears similar to that described recently for cryptococcal fungistatic cell wall-binding mAbs (Rodrigues et al., 2000).
The above results presuppose that melanin formation occurs in vivo. Early studies suggested that melanin was present based on staining of C. neoformans cells in tissue with Masson-Fontana stain, however, this technique is not specific for melanin as both in vitro melanized and non-melanized cells stain positively (Kwon-Chung et al., 1981). However, histological studies of human tissues have shown that cryptococcal cells sometimes appeared dark (Lee et al., 1996) and murine studies have demonstrated a progressive darkening and thickening of the cell wall during infection (Nosanchuk et al., 1999a; Feldmesser et al., 2001). Evidence for laccase activity in vivo was provided by the identification of the specific melanin degradation products pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4-tricarboxylic acid (PTCA) in cryptococcal cells isolated from infected tissue (Liu et al., 1999b). However, the investigators did not detect evidence for polymerization of melanin (Liu et al., 1999b). Following the demonstration that fungal melanin is immunogenic (Nosanchuk et al., 1998), mAbs to melanin were generated and shown to react with C. neoformans cell walls in infected tissue (Fig. 2) (Rosas et al., 2000b). Melanin-binding phage also labelled cryptococcal cell walls in tissue (Nosanchuk et al., 1999b). Furthermore, murine infections with C. neoformans stimulated a brisk antibody response to fungal melanin, which provided immunological proof for the synthesis of the pigment in vivo (Nosanchuk et al., 1999b).
However, these findings did not conclusively establish that polymerized melanin was formed in vivo because it was possible that antibody was binding to melanin precursors that were also immunogenic. Direct evidence for the polymerization of melanin in vivo was demonstrated by the isolation of melanin particles (‘ghosts’) from infected rodent tissues by serial treatment with enzymes, denaturant, and hot acid (Fig. 3) (Rosas et al., 2000b). The particles recovered were similar in size and shape as C. neoformans cells and were labelled by melanin-specific mAb (Rosas et al., 2000b). No melanin particles were identified from mice infected with laccase-negative mutants (Rosas et al., 2000b). These techniques were also applied to study infected human brain tissue. The melanin-binding mAb and phage both labelled cryptococcal cells in tissue and melanin particles were recovered from tissue treated with enzymes, denaturant and acid that reacted with the melanin-binding reagents (Nosanchuk et al., 2000). Taken as a whole, the data show that polymerized melanin is formed during infection.
Using the protocols developed for demonstrating the presence of polymerized melanin production in C. neoformans, melanization was recently demonstrated in both conidia and yeast forms of the dimorphic pathogens P. brasiliensis (Gómez et al., 2001) and H. capsulatum (Nosanchuk et al., 2002). Additionally, both pathogens produced melanin during murine infection. This technique has also been applied to identify melanin in the conidial form of Penicillium marneffei (Hamilton and Gomez, 2002). However, future studies are required to define whether an association between melanization and virulence exists for these pathogens.
Although less extensively studied, melanin production has also been linked with virulence in the human fungal pathogens E. dermatitidis and members of the genus Aspergillus. In E. dermatitidis, melanin-deficient mutants displayed reduced virulence compared to wild-type strains (Dixon et al., 1987; 1992) and the ability to produce invasive hyphal forms in the mutants was severely limited (Dixon et al., 1989). Disruption of the putative polyketide synthase gene in E. dermatitidis resulted in albino cells with attenuated virulence (Feng et al., 2001). Albino mutants of A. fumigatus are less lethal in murine infection than melanized, wild-type strains (Jahn et al., 1997). In A. fumigatus, disruption of the alb1 gene encoding a putative polyketide synthase results in strains with reduced virulence to mice compared to strains with intact expression of the polyketide synthase (Tsai et al., 1998). Additionally, a study comparing melanizing and non-melanizing strains of Basidiobolus sp. found that the melanized isolates were associated with human disease (Cutler and Swatek, 1969).
Association of melanin with fungal virulence in plant infection
In contrast to plant infection, no clear association between melanization and generation of invasive hyphae in animal or human infection has been demonstrated. In a murine infection model, significantly more invasive hyphal growth was noted for melanized E. dermatitidis hyphae compared to a melanin-deficient mutant strain (Dixon et al., 1991). Subsequent in vitro experiments also showed that E. dermatitidis hyphal penetration of agar was dependent on melanin production (Brush and Money, 1999). This data suggest that as in plant infection, melanin synthesis may be involved in hyphal invasion in tissue.
Mechanisms by which melanin contributes to virulence (Fig. 4)
Protection against oxidants
Melanins have a strong affinity for metals, are highly effective scavengers of free radicals (Sichel et al., 1991), and have electron transfer properties (Gan et al., 1976). Electron transfer from free radical species generated in solution to C. neoformans melanin has been demonstrated by ESR spectroscopy (Wang et al., 1995). Cryptococcus neoformans melanin is involved in the reduction of Fe3+ to Fe2+ (Nyhus et al., 1997) and the pigment can facilitate redox cycling by maintaining the reducing capacity of extracellular redox buffers through the exportation of electrons to form extracellualr Fe2+ (Jacobson and Hong, 1997). The antioxidant function of C. neoformans melanin may be particularly important at 37°C where the activity of superoxide dismutase is reduced (Jacobson et al., 1994). A. niger melanin is also capable of functioning as a redox buffer. Measurements of ESR signal initially increase and then decrease during progressive reduction of A. niger melanin and during subsequent re-oxidization of the compound (Lukiewicz et al., 1980).
Protection against oxidative injury in pigmented C. neoformans cells was first linked to catecholamine utilization for the production of melanin (Polacheck et al., 1990; Jacobson and Emery, 1991b). Melanized C. neoformans were subsequently shown to be less susceptible to killing by hypochlorite and permanganate (Jacobson and Tinnell, 1993). Albino mutants were more sensitive to hyperoxia (Emery et al., 1994). Melanized cryptococcal cells were also more resistant to oxygen- and nitrogen-derived radicals than non-melanized cells (Wang and Casadevall, 1994c; Wang et al., 1995). DHN melanin in E. dermatitidis and A. alternata protects against permanganate and hypochlorite (Jacobson et al., 1995). As with C. neoformans (Jacobson and Tinnell, 1993), melanin did not limit the effects of H2O2, a weak fungicide, on E. dermatitidis cells (Jacobson et al., 1995). Pigmented E. dermatitidis were protected against neutrophil oxidative burst due to polymerized melanin, and not carotenoids or the melanin precursor scytoalone (Schnitzler et al., 1999). Albino mutant Sporothrix schenckii isolates are more susceptible to killing by nitrogen- or oxygen-derived radicals (Romero-Martinez et al., 2000). Albino mutants of A. fumigatus are more sensitive to killing by reactive oxygen compounds and the reactive oxidants were quenched by melanized cells (Jahn et al., 1997). Reactive oxygen compounds secreted by infected plants are toxic to melanin-deficient mutants of M. grisea (Aver’yanov et al., 1989).
Proteus mirabilis (Agodi et al., 1996) and Burkholderia cepacia (Nelson et al., 1994; Zughaier et al., 1999) are clinically important bacteria that produce melanin. Melanin acts as a free radical scavenger in P. mirabilis (Agodi et al. (1996). Burkholderia cepacia melanin protects the microbe from superoxide anion insult (Zughaier et al., 1999). The capacity of B. cepacia to produce melanin has been proposed as a significant factor in its colonization and transmission (Zughaier et al., 1999). Azospirillum chroococcum melanin has been associated with protection against toxic oxygen compounds (Shivprasad and Page, 1989) and iron binding by melanin in Azospirillum salinestris may protect against damage from hydrogen peroxide (Page and Shivprasad, 1995). These observations suggest that melanin contributes to virulence by protecting melanized fungal and bacterial cells in tissue against the oxidative burst of activated host effector cells.
Effect on phagocytosis and phagocytic killing
Melanized C. neoformans cells are more resistant to antibody-mediated phagocytosis than melanin-deficient cells (Wang et al., 1995). Phagocytosis can be altered by the surface hydrophobicity and cell charge of C. neoformans (Kozel, 1983). The mechanism by which melanization interferes with phagocytosis is unknown as it does not significantly alter surface hydrophobicity of encapsulated cells (Wang et al., 1995). However, melanins are charged polymers (White, 1958) and the cell charge of C. neoformans may be significantly altered by the presence of melanin, particularly if the polysaccharide capsule is small or absent (Nosanchuk and Casadevall, 1997). In addition to reducing phagocytosis, melanization protects C. neoformans against killing by macrophages (Wang et al., 1995). Comparison of the macrophage killing activity for 18 C. neoformans strains that varied in melanin synthesis revealed an inverse correlation between melanization capacity and susceptibility to macrophages killing (Fig. 5). Melanization of S. schenckii conidia enhances resistance to phagocytosis and killing by human monocytes and murine macrophages (Romero-Martinez et al., 2000). Although phagocytosis by neutrophils of E. dermatitidis is not affected by the presence or absence of melanin, melanized cells are significantly protected against killing (Schnitzler et al., 1999). Protection against killing by neutrophils also occurs with several other melanotic species of Exophiala (De Hoog et al., 2000). In addition to reactive oxygen species, phagocytic cells can produce defensins and other microbicidal peptides and melanized C. neoformans are less susceptible to the toxic effects of such compounds compared to non-melanized cells (Doering et al., 1999). The mechanism of action in this case appears to be absorption of the microbiocidal peptide such that it interferes with the peptide reaching its target. The reduction in killing in pigmented cells is likely due to the capacity of melanin to reduce their susceptibility against oxidative and non-oxidative injury.
Resistance to antimicrobial compounds
Melanin is known to bind diverse compounds (Kaliszan et al., 1993; Larsson, 1993). This capability has led to interesting insights into the pathogenesis of diverse diseases, such as Parkinson's disease. In Parkinson's disease there is a loss of pigment in the melanotic dopaminergic neurons in the substantia nigra of the brain. A tantalizing connection between melanin and the etiology of Parkinson's disease comes from the observation that heroin contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) caused similar neurological disease in drug users, possibly because the drug binding properties of melanin concentrated this compound in the neurons in the substantia nigra (Herrero et al., 1993).
Amphotericin B is a potent broad-spectrum antifungal that is recommended for the treatment of life-threatening cryptococcosis (Saag et al., 2000). Growth of C. neoformans in the presence of L-DOPA provides significant protection against amphotericin B (Wang and Casadevall, 1994b). Further studies confirmed this finding and showed that melanization also protects C. neoformans against the activity of caspofungin, but not fluconazole, itraconazole, or flucytosine (Van Duin et al., 2002). Similar results were obtained using melanized and non-melanized H. capsulatum yeast cells (Van Duin et al., 2002). Elemental analysis revealed that the C : N : O ratio of melanin particles exposed to amphotericin B or caspofungin was altered, which is consistent with the absorption of those drugs by melanin (Van Duin et al., 2002). No change in elemental composition occurred with the incubation of the other antifungal agents. Hence, the data suggest that cell wall cryptococcal melanin binds amphotericin B and caspofungin and prevents them from reaching their target sites. This observation suggests a potential explanation for the difficulty in eradicating C. neoformans with amphotericin B (Zuger et al., 1986). For caspofungin, this finding explains why the drug is active in vitro against non-melanized cells (Franzot and Casadevall, 1997), but is ineffective in experimental infection with C. neoformans (Abruzzo et al., 1997) or H. capsulatum (Kohler et al., 2000). Interestingly, no differences were detected by standard MIC testing between melanized and non-melanized C. neoformans (Van Duin et al., 2002). This appeared to be due to the lack of melanin precursors in the medium used for the MIC or to an inherent insensitivity of this method to detect the effect (Van Duin et al., 2002). Similarly, no differences in susceptibility to antifungals were noted between albino and pigmented E. dermatitidis (Polak and Dixon, 1989). In contrast, trifluoperazine has increased toxicity for melanized C. neoformans cells (Wang and Casadevall, 1996b). The capacity of melanin to bind some drugs suggests a potential mechanism for the observation that phenothiazines have activity against C. neoformans (Eilam et al., 1987).
There is little data on the function of melanin against fungicides in plant pathogens. In M. fructicola, melanin appears to protect against dicarboximide (Benes and Ritchie, 1984). Infection of grapevines by Botrytis cinerea may actually be inhibited by the conversion of the grapevine's metabolite resveratrol by laccase into secondary compounds toxic to the fungus (Schouten et al., 2002). Pseudomonas aeruginosa melanin does not appear to serve a protective role against antibacterial agents in vitro (Rozhavin, 1978; Rozhavin and Sologub, 1979). Melanin in Bacillus thurigiensis may protect the bacteria against pesticides (Patel et al., 1996).
Melanins are immunogically active compounds. Injection of purified cryptococcal melanin into mice results in the generation of a brisk antibody response (Nosanchuk et al., 1998). The development of a solid-phase ELISA utilizing melanin facilitated this work (Rosas et al., 2000a) and was also used in the identification of melanin-binding mAbs (Rosas et al., 2000b). Furthermore, antibodies to melanin develop during experimental cryptococcosis (Nosanchuk et al., 1999b). The antibodies generated to melanin during infection may be protective, but given the association with melanin and autoimmune diseases, such as vitiligo, the antibody response could conceivably be deleterious in some circumstances. Melanin from C. neoformans activates the alternative complement cascade (Rosas et al., 2002). Grape melanin synthesized by a tyrosinase has anti-inflammatory and immunomodulatory properties (Avramidis et al., 1998). Administration of grape melanin inhibited the development of primary and secondary adjuvant induced disease (AID) in rats and significantly reduced the levels of IL-1β IL-6 and TNF-α in these animals (Avramidis et al., 1998). Similarly, synthetic melanin inhibited production of IL-1β, IL-6, IL-10 and TNF-α in human peripheral monocytes that were stimulated with lipopolysaccharide (Mohagheghpour et al., 2000). Interestingly, incubation of J774 macrophages with the melanin precursor 5,6-dihydroxyindole-2-carboxylic acid results in increased generation of nitric oxide following lipopolysaccharide stimulation (D’Acquisto et al., 1995).
Injection of melanin particles isolated from C. neoformans into the peritoneal cavity of mice resulted in the formation of granulomas in the liver, spleen and lung (Rosas et al., 2002). Granulomatous inflammation occurred around melanin particles and resembled a foreign body reaction (Fig. 6) (Rosas et al., 2002). These observations reveal that fungal melanin can elicit a vigorous inflammatory reaction in healthy mice, presumably because melanin behaves as a foreign substance that cannot be broken down by host enzymes. During infection with C. neoformans, the inflammatory reaction is less intense due to inhibitory effects of the capsular polysaccharide that diffuses into tissues (Vecchiarelli, 2000). Nevertheless, C. neoformans cells in chronic and latent infections are usually encased in well formed granulomas similar to those observed with melanin particles (Goldman et al., 2000). Hence, the indigestible and highly reactive material in the cell walls of the yeast cells in tissue may serve as a potent immunomodulator during infection. In fact, many of the fungal pathogens that are notoriously associated with latency and granuloma formation make melanin. Hence, it is conceivable that cell-wall associated melanin in tissue provides an indigestible material that serves as a ‘foreign-body’-like material that interferes with clearance of infection while at the same time stimulating intense inflammation.
Bacillus subtilis is a spore-forming bacterium that produces a protein coat that contains a brown pigment around the endospore (Schaeffer, 1969). The spore coat protein CotA is a classical laccase which appears to catalyse the synthesis of melanin (Hullo et al., 2001). The spore coat of B. subtilis confers resistance to UV light and hydrogen peroxide (Riesenman and Nicholson, 2000) and pigmented strains are dramatically more protected against injury compared to lightly pigmented strains (Hullo et al., 2001). The related bacterium, Bacillus thuringiensis also produces a melanin (Hoti and Balaraman, 1993) that protects the microorganism from UV damage and pesticides (Patel et al., 1996). Laccase-like activity has also been demonstrated in Bacillus sphaericus (Claus and Filip, 1997). In P. aeruginosa, melanization protects against UV irradiation and high or low concentrations of oxygen (Rozhavin, 1983).
Melanin synthesis meets rigorous criteria for definition as a virulence factor in C. neoformans (Casadevall & Pirofski 1999; 2001), and this example provides a paradigm for understanding the contribution of melanogenesis to microbial pathogenesis. The fact that C. neoformans melanin-deficient strains are hypovirulent but viable meets the definition of virulence factor as a component needed for host damage, but not viability. Melanin presumably contributes to microbial virulence by promoting survival in a host and thus allowing the organism to cause disease. For C. neoformans cellular melanization has been associated with reduced susceptibility to a variety of insults (Fig. 4) and it is likely that this paradigm is applicable to other melanotic organisms. However, melanin can also elicit intense inflammatory responses that may result in host damage. Although the association of melanization and virulence and the widespread synthesis of melanin pigments by diverse pathogens insures its importance in the field of microbial pathogenesis, it is worthwhile to note that this pigment system presents major unsolved problems in the fields of cell biology, enzymatic synthesis and degradation, and structural biology. Melanotic fungi are able to deposit melanin in their cell walls and remodel the pigment during growth through mechanisms that are not understood. Clearly, cell division and morphogenesis must be tightly regulated in conjunction with melanin synthesis and degradation for cells to grow and change shape. Currently, relatively little is known about the process by which pigmented cells remodel their melanin layers. Hindering progress is the fact that the structure of melanin lies beyond our current analytical capabilities, as the available techniques are inadequate to provide a structural solution for melanin. However, we are hopeful that the importance of this system for fungal, bacterial and helminthic pathogenesis will provide a strong impetus for additional research in this enigmatic field.
A. Casadevall was supported by National Institute of Health (NIH) grants AI33774, AI13342 and HL59842. J. D. Nosanchuk was supported by NIH grant AI01489. We thank J. Steenbergen for editorial assistance.