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Abstract
Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.
Highlights
Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections
Upon light microscopy examination of melanized cells from two isolates representing the C. neoformans and C. gattii species complexes, H99 and R265, respectively (Table 1), we observed that a subset of C. gattii R265 cells displayed highly pigmented cell walls, which were not observed for C. neoformans H99 (Fig. 1)
We tested the generality of cell-wall ultrastructural characteristics associated with melanin deposition by using transmission EM to further compare the melanized cell walls of C. neoformans H99 with C. gattii R265, which is a strain from the VGIIa molecular subtype reported to exhibit robust melanin production at 37 °C (44 –46)
Summary
MAT␣ C. neoformans C. neoformans MAT␣ csr2::T-DNA bearing a frameshift mutation in CSR2 MAT␣ C. gattii C. gattii MAT␣ chs3::T-DNA (NEO). Melanins are complex heterogeneous polymers of phenolic and/or indolic origin characterized by a dark color, insolubility in most solvents, and the possession of a stable free radical signature [23, 24] These natural pigments are synthesized by members of all biological kingdoms and have a wide array of functions. The identity of the melanin precursor (norepinephrine, epinephrine, methyl-LDOPA, L-DOPA) alters the pigment structure as well as the polysaccharide- and lipid-based cellular scaffold associated with its deposition [32, 33]. These analyses dovetail with previous reports in other fungi (34 –36) as well as in insects [37],
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