Cryptococcus neoformans is a pathogenic fungus that produces melanin when incubated in the presence of certain phenolic substrates such as L-3,4-dihydroxyphenylalanine (L-dopa). Melanin is an enigmatic polymer that is deposited in the cell wall and contributes to virulence. Substantial progress has been made in understanding the synthesis of melanin and the mechanisms by which it contributes to virulence, but relatively little is known about how melanin is rearranged during growth and budding. In this study we used transmission and scanning electron microscopy and immunofluorescence of melanized cells and melanin 'ghosts' to study the process of melanization during replication. Budding in melanized C. neoformans results in focal disruption of cell-wall melanin at the bud site. In the presence of L-dopa, bud-related melanin defects are repaired and daughter cells are melanized. However, in the absence of substrate, mother cells cannot repair their melanin defects and daughter cells are non-melanized. Hence, melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo in buds. These results imply that melanin remodelling occurs during cell growth in a process that involves degradation and synthesis at sites of budding.
INTRODUCTIONCryptococcus neoformans is an encapsulated, environmental fungus that can cause life-threatening meningitis, particularly in immunocompromised individuals. The ability of C. neoformans to make melanin was discovered in the early 1960s (Staib, 1962(Staib, , 1963 and was associated with virulence in the 1980s Rhodes et al., 1982). Melanins are rigid, acid-resistant polymers of uncertain structure that are found in all biological kingdoms (Hill, 1992). The synthesis of melanin by C. neoformans occurs only in the presence of phenolic compounds, such as L-3,4-dihydroxyphenylalanine (L-dopa), and is catalysed by a laccase Williamson, 1994). The C. neoformans laccase has recently been shown to be tightly associated with the cell wall by a hydrolysable bond (Zhu et al., 2001). When the fungus grows in the presence of phenolic compounds, melanin is deposited in the cell wall (Wang et al., 1995) and comprises approximately 15 % of the dry weight of late-stationary-phase melanized C. neoformans (Wang & Casadevall, 1996). C. neoformans is melanized in the environment (Nosanchuk et al., 1999b) and during human infection (Nosanchuk et al., 2000). In the environment melanin is thought to protect the fungus against extremes of temperature (Rosas & Casadevall, 1997), ultraviolet light (Wang & Casadevall, 1994a), heavy metals (Garcia-Rivera & Casadevall, 2001) and amoeboid predators (Steenbergen et al., 2001). During infection melanin appears to function in virulence by protecting fungal cells against microbicidal oxidants (Wang & Casadevall, 1994b) and peptides (Doering et al., 1999) produced by immune effector cells. Furthermore, melanin may interfere with the development of effective cellmediated responses (Huffnagle et al., 1995) and could affect the activation ...