Vesicle-associated melanization in Cryptococcus neoformans

Abstract

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. Acidgenerated 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.

The effect of L-DOPA onCryptococcus neoformansgrowth and gene expression

“…The paucity of gene responses is also consistent with our recent proposal that melanin is synthesized in vesicles that are subsequently exported to the cell wall 41 since this model involves pigment synthesis from highly reactive and toxic intermediates of L-DOPA oxidation in a confined membrane enclosed compartment that shields cellular processes. A similar strategy for avoiding melanin precursor toxicity is used by mammalian cells, which make melanins within melanosomes.…”

Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins

“…Similarly, our group observed B. anthracis vesicle structures of comparable size that are spherical with bilayered membranes and electron-dense luminal contents. Light scattering analysis revealed a heterogenous population of vesicle diameter that was consistent with the size predicted from electron microscopic studies, with the caveat that these techniques overestimate and underestimate vesicle diameters (24,37). We note with interest that the dimensions of Gram-positive, Gram-negative, and fungal vesicles are similar, despite a very high likelihood that the mechanisms of vesicle production differ markedly among these phylogenetically, distant microbes, which differ in cell-wall architecture.…”

“…The effective diameter and size distribution of vesicles preparations in 10 mM dextrose were measured using a 90Plus/BI-MAS Multi Angle Particle Sizing analyzer (Brookhaven Instruments Corp.), as described (37).…”

Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

“…Secreted vesicles exhibit urease, phosphatase, and laccase activity (15,16), so we tested the vesicle samples obtained from different centrifugation times for all three enzymatic activities. The highest specific activity for all three assays was in the 5-min and 60-min samples, but these differences were not significant (Fig.…”