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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.

Cryptococcus neoformans is subject to oxidative attack by host immune cells; consequently, oxidant-resistant mechanisms may be important in pathogenesis. Mutations at the OXY2 locus confer decreased laccase and increased sensitivity to hyperbaric oxygen in the background of the oxyl mutation, but, alone, do not confer sensitivity to oxidants. Because metal deficiency can potentiate or ameliorate sensitivity to oxidants, and because the melanin-synthesizing laccase contains copper, we investigated copper acquisition in an oxy2 mutant. We found that its external Cu/Fe reductase activity was lower than that of wild type, and although copper deprivation induced the reductase in the wild type, it did not do so in oxy2. Oxy2 is sensitive to copper chelation but resistant to high copper, suggesting that copper transport is decreased. The strain expresses large amounts of alternate oxidase in response to Cu-chelation, perhaps in response to defective, Cu-deprived cytochrome oxidase, and is resistant to the oxidant, plumbagin, under this condition, perhaps due to the high alternate oxidase. These phenotypes are similar to those of the mac1- mutant of Saccharomyces cerevisiae and the melanin-deficient grisea mutant of Podospora anserina, in which homologous transcriptional activators for the reductase and copper transporter genes are mutated. They constitute physiologic evidence that oxy2 is mutated in a homologous copper-related transcriptional activator of C. neoformans.

Cryptococcus neoformans secretes 3-hydroxyanthranilate (3HAA), but the utility is unknown. Exogenous 3HAA promoted growth of cultures starved for iron with transferrin, presumably by releasing Fe(III) reductively. Exogenous 3HAA protected C. neoformans from strong oxidants, suggesting a role in resistance to killing by immune cells. 3HAA represents an endogenous laccase substrate, in that crude laccase preparations convert 3HAA to cinnabarinic acid, whereas 3HAA concentrations are higher in Lac- mutants. We isolated hypersecreting mutants as highly fluorescent clones. Because C. neoformans has been isolated from rotting wood, we looked for a role in degradation of lignin. Using cyclic voltammetry, we found no electrochemical evidence that organic oxidation products of 3HAA are capable of oxidizing lignin. We found neither cellulose dehydrogenase nor lignin peroxidase enzymic activity, nor did C. neoformans grow on cellulose as carbon source. We found no evidence for production of Fenton reagent by cultures, even in the presence of transition metal ions or of those and 3HAA. The biological utility of 3HAA may be related to its functions as reducing agent and, conceivably, as laccase substrate. It does not appear to attack wood, nor does C. neoformans appear to have a mechanism to rot wood.

Cryptococcus neoformans is a pathogenic basidiomycete that causes meningitis in immunocompromised patients. In this paper, we demonstrate that a previously described oxidant-sensitive mutant, oxy1, has constitutive ferric reductase and iron uptake, similar to a ferric reductase regulatory mutant, frr1. Through meiotic genetic analysis, we show that the two mutations are allelic. By complementation of frr1 with a genomic library, we isolated a gene, MRS3/4. The encoded protein is a putative solute transporter of the inner mitochondrial membrane. Disruption of this gene led to high ferric reductase, iron uptake and iron content, as well as increased sensitivity to hydrogen peroxide and slow growth in low iron medium. The disrupted gene is allelic with oxy1 and frr1. We sequenced the oxy1 and frr1 alleles of MRS3/4 and found that the frr1 mutation results in a premature stop codon, while the oxy1 mutation results in the substitution of a highly conserved glutamate residue with lysine. The Mrs3/4 protein appears to be involved in mitochondrial iron transport in eukaryotes. Resistance to strong oxidants requires stringent control of iron metabolism.

Iron is tightly controlled in mammalian tissues and regulates virulence factors in various pathogenic organisms. The influence of Fe availability upon production of cryptococcal capsular polysaccharide was studied. Polysaccharide, measured as cell-bound glucuronyl residues, increased more than threefold as available Fe in the culture medium was varied from repletion to tight sequestration and depletion in five incremental steps. Since physiologic CO2 concentration may serve as stimulus for cryptococcal polysaccharide synthesis, the combined effect of Fe availability and CO2 on encapsulation was studied. Addition of dissolved, loosely chelated Fe moderated the effect of CO2. Tight chelation of dissolved Fe potentiated the CO2 effect. Tissue from infected mice showed heavily encapsulated organisms, consistent with results with physiologic CO2 concentration and Fe deprivation. In conclusion, cryptococcal polysaccharide synthesis is increased by limitation of ferric iron availability to the cell and by dissolved CO2, and the two effects are additive.

The clinical presentation and autopsy findings in the case of a patient with acute myelomonocytic leukemia and trichinosis are described. Cellular immunity is an important host defense against nematode infections such as strongyloidiasis. It is therefore probable that the severe trichinosis seen in this immunosuppressed patient was more than coincidental. Due to concomitant failure of humoral immunity, serodiagnosis of trichinosis would have been impossible. The only means of making the diagnosis antemortem would have been muscle biopsy.