Budding of melanized Cryptococcus neoformans in the presence or absence of l-dopa

Abstract

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 ...

Impact of Melanin on Microbial Virulence and Clinical Resistance to Antimicrobial Compounds

“…Similarly, no differences in susceptibility to antifungals by MIC assays were measured between albino and pigmented cells of E. dermatitidis (101), H. capsulatum (124), or Blastomyces dermatitidis (96). However, the growth of melanized C. neoformans yeast cells in medium without a phenolic substrate resulted in large defects in the melanin layer of the parent cells after budding and an absence of melanin in the daughter cells (89). Hence, even if melanized cells are used initially, the daughter cells lack melanin, and the CLSI methodology does not compare the susceptibilities of melanized and nonmelanized cells.…”

Detection of Antibodies against Paracoccidioides brasiliensis Melanin inIn VitroandIn VivoStudies during Infection

“…Moreover, melanin pigments from P. brasiliensis yeast cells are synthesized during mammalian infection, and melanin ghosts can be isolated from P. brasiliensis-infected tissues (11). The ability of conidia to produce melanin-like compounds in the absence of substrates such as L-DOPA differentiates the melanization process seen in P. brasiliensis mycelia from that seen in C. neoformans, a fungus that relies on the presence of an exogenous substrate to drive the process (29,35). The present work has focused on the generation of MAbs to P. brasiliensis melanin.…”

“…The paraffin-embedded pulmonary tissues were passed through gradients of decreasing xylol and alcohol (100 to 50%) solutions before initiating the process. Frozen samples were then transferred to a Leica EM AFS freeze substitution unit (Leica Microsystems, Vienna, Austria), and ultrathin lung tissue sections were placed on nickel grids and processed as described previously (29).…”

Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors

“…Although phospholipase and phenoloxidase activity are clearly linked to virulence, and melanin can be detected within human brains and other tissues infected with C. neoformans (Casadevall et al, 2000;Chen et al, 1997;Cox et al, 2001;Jacobson & Emery, 1991;Kwon-Chung et al, 1982;Kwon-Chung & Rhodes, 1986;Nosanchuk & Casadevall, 2003;Noverr et al, 2004;Rhodes et al, 1982;Salas et al, 1996;Williamson, 1997), the levels of phospholipase and phenoloxidase activity have not been shown to be associated with virulence. Like the overwhelming majority of clinical isolates, all our isolates exhibited phospholipase and phenoloxidase activity Chen et al, 1997).…”