Effect of melanization upon porosity of the cryptococcal cell wall

The cell-wall constituent, melanin, is a virulence factor for pathogenic fungi, but its structural and mechanistic role is not clearly understood. As intermediates in melanin formation are cross-linking agents, we wondered whether melanized cell walls might be more highly cross-linked and less porous than non-melanized cell walls. The fungal pathogen Cryptococcus neoformans makes melanin only in the presence of exogenous catechols; we cultivated it with and without 1 mmol/l dopamine. We prepared mechanically intact melanized and non-melanized cell walls by boiling cells in 10% sodium dodecyl sulfate; electron microscopy showed disruption of cytoplasm. We poured the resulting spheres into columns and studied the elution behavior of graded dextrans. High-molecular-weight dextrans eluted earlier than low-molecular-weight dextrans, which, in turn, eluted before glucose, behavior characteristic of size-exclusion chromatography. We calculated the thresholds above which the polymers were totally excluded from the cell walls. Melanized cells exhibited a threshold of molecular weight 30 600, non-melanized, 270 000 (P <0.01). The corresponding Einstein-Stokes radii are 4.0 and 10.6 nm, respectively; these represent the calculated largest pore sizes for each condition. We conclude that melanized cell walls are considerably less porous than non-melanized cell walls.