The role of melanins in melanotic fungi for pathogenesis and environmental survival.

Melanins provide fungi protection from environmental stressors, support their ecological roles, and can confer virulence in pathogens. While the function, structure, and synthesis of melanins in fungi are not fully understood, they have been shown to have varied roles. Recent research has revealed a wide range of functions, from radiation resistance to increasing virulence, shedding light on fungal diversity. Understanding fungal melanins can provide useful information, from harnessing the properties of these various melanins to targeting fungal infections.Key Points• Melanotic fungi are widespread in nature. • Melanin functions to protect fungi in the environment from a range of stresses. • Melanin contributes to pathogenesis and drug resistance of pathogenic fungi.

Reduced virulence of melanized Cryptococcus neoformans in Galleria mellonella.

Fungal melanins are important in the virulence of many pathogenic fungi. In this study, we examined the role of melanin in the interaction between Cryptococcus neoformans and the invertebrate host, Galleria mellonella. C. neoformans was able to melanize in the presence of G. mellonella homogenate, indicating the presence of melanin substrates. Melanization was confirmed by the recovery of acid-resistant particles that were recognized by anti-melanin antibodies. In addition, we tested the effect of fungal melanization on virulence. Surprisingly, G. mellonella larvae infected with melanized fungal cells lived longer than those infected with non-melanized fungi. When the cellular immune response of G. mellonella to melanized and non-melanized cells was compared, inflammatory nodules were observed in both groups. However the response was stronger in larvae infected with melanized cells. These results suggest that fungal melanin activates the immune response of G. mellonella, thereby resulting in the decreased virulence observed with melanized cells.

Synthesis and assembly of fungal melanin.

Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from L-3,4-dihydroxyphenylalanine (L-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.

The effect of L-DOPA on Cryptococcus neoformans growth and gene expression.

Cryptococcus neoformans is unusual among melanotic fungi in that it requires an exogenous supply of precursor to synthesize melanin. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. L-3,4-dihydroxyphenylalanine (L-DOPA) is a natural catecholamine that is frequently used to induce melanization in C. neoformans and L-DOPA-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents and heavy metals. Given that C. neoformans needs exogenous substrate for melanization one question in the field is the extent to which melanin-associated phenotypes reflect the presence of melanin or metabolic changes in response to substrates. In this study we analyze the response of C. neoformans to L-DOPA with respect to melanization, gene expression and metabolic incorporation. Increasing the concentration of L-DOPA promotes melanin formation up to concentrations > 1 mM, after which toxicity is apparent as manifested by reduced growth. The timing of C. neoformans cells to melanization is affected by growth phase and cell density. Remarkably, growth of C. neoformans in the presence of L-DOPA results in the induction of relatively few genes, most of which could be related to stress metabolism. We interpret these results to suggest that the biological effects associated with melanization after growth in L-DOPA are largely due to the presence of the pigment. This in turn provides strong support for the view that melanin contributes to virulence directly through its presence in the cell wall. 

Vesicle-associated melanization in Cryptococcus neoformans.

Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor L-3,4-dihydroxyphenylalanine (L-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled L-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.

Cryptococcus neoformans laccase catalyses melanin synthesis from both D- and L-DOPA.

The human fungal pathogen Cryptococcus neoformans produces melanin in the presence of various substrates, including the L enantiomer of 3,4-dihydroxyphenylalanine (DOPA). The enzyme laccase catalyses the formation of melanin by oxidizing L-DOPA, initiating a series of presumably spontaneous reactions that ultimately leads to the polymerization of the pigment in the yeast cell wall. There, melanin protects the cell from a multitude of environmental and host assaults. Thus, the ability of C. neoformans to produce pigments from a variety of available substrates is likely to confer a survival advantage. A number of C. neoformans isolates of different serotypes produced pigments from D-DOPA, the stereoisomer of L-DOPA. Acid-resistant particles were isolated from pigmented C. neoformans cells grown in the presence of D-DOPA. Biophysical characterization showed the particles had a stably detectable free-radical signal by EPR, and negative zeta potential, similar to L-DOPA-derived particles. No major differences were found between L- and D-DOPA ghosts in terms of binding to anti-melanin antibodies, or in overall architecture when imaged by electron microscopy. C. neoformans cells utilized L- and D-DOPA at a similar rate. Overall, our results indicate that C. neoformans shows little stereoselectivity for utilizing DOPA in melanin synthesis. The ability of C. neoformans to use both L and D enantiomers for melanization implies that this organism has access to a greater potential pool of substrates for melanin synthesis, and this could potentially be exploited in the design of therapeutic inhibitors of laccase.

New insights on the pathogenesis of invasive Cryptococcus neoformans infection.

Disseminated cryptococcosis begins with infection of the lungs via inhalation. This is followed by escape from the lungs and entry into the bloodstream allowing dissemination to the brain and central nervous system. We discuss the steps involved in dissemination and the host and microbial factors that influence each step. For the host, containment in the lung is accomplished with a combination of cell-mediated and antibody responses. Dissemination occurs when these systems fail and/or when phagocytic cells that fail to kill the yeast instead act as a niche for replication. One of the main microbial factors affecting dissemination is the polysaccharide capsule, a major virulence factor that promotes dissemination at every step. Secreted enzymes are important, including laccase and phospholipase B, which promote escape from the lungs, and urease, which contributes to crossing the blood-brain barrier. Lastly, a number of regulatory factors contribute, especially to growth of Cryptococcus neoformans in the brain.

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates.

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in L-dopa, methyl-dopa, (-)-epinephrine or (-)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by L-dopa. Electron microscopy indicated that L-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans.

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.

Activation of pleiotropic drug resistance by the J-protein and Hsp70-related proteins, Zuo1 and Ssz1.

Ssz1 (Pdr13) and Zuo1, members of the Hsp70 and J-protein molecular chaperone families, respectively, form a heterodimer and function on the ribosome with the Hsp70, Ssb, presumably assisting folding of newly synthesized polypeptides. As it has also been reported that Ssz1 induces pleiotropic drug resistance (PDR) when overexpressed, a possible role for Zuo1 in PDR was investigated. The C-terminal domain of Zuo1, which is dispensable for Zuo1's chaperone function on the ribosome, is both necessary and sufficient for PDR induction by Zuo1. A single domain of Ssz1, the N-terminal ATPase domain, is sufficient for PDR induction as well, indicating that Ssz1 does not function as a chaperone in PDR. No role for Ssb was found in PDR; overexpression did not affect PDR, nor was its presence required for Ssz1's or Zuo1's effect on PDR. As our results also indicate that Ssz1 and Zuo1 must be free of ribosomes to induce PDR, we propose that Ssz1's and Zuo1's function in PDR is distinct from their role as ribosome-associated co-chaperones and may be regulatory in nature.

Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding?

Folding of many cellular proteins is facilitated by molecular chaperones. Analysis of both prokaryotic and lower eukaryotic model systems has revealed the presence of ribosome-associated molecular chaperones, thought to be the first line of defense against protein aggregation as translating polypeptides emerge from the ribosome. However, structurally unrelated chaperones have evolved to carry out these functions in different microbes. In the yeast Saccharomyces cerevisiae, an unusual complex of Hsp70 and J-type chaperones associates with ribosome-bound nascent chains, whereas in Escherichia coli the ribosome-associated peptidyl-prolyl-cis-trans isomerase, trigger factor, plays a predominant role.