Ferrous Iron Uptake in Cryptococcus neoformans

Iron is required for oxidoreductase enzymes in almost all living organisms. Although iron is widely distributed in the environment, under neutral pH and aerobic conditions oxidation of iron to poorly soluble Fe(III) causes ambient concentrations to be very low. Microbes mobilize environmental iron for uptake according to one of three basic strategies: by secreting iron-chelating chemicals, by reducing insoluble environmental Fe(III) to soluble Fe(II), or by expressing receptors and deferration mechanisms for iron-containing proteins. The most widely studied microbial iron uptake systems use secreted hydroxamate or catecholate Fe(III) chelators, termed siderophores, and cell surface receptors for the ferrated complex (21). One such siderophore, deferoxamine (Desferal; Ciba-Geigy), is manufactured by fermentation and is used clinically to mobilize trivalent metals. Saccharomyces cerevisiae (16), Listeria monocytogenes (5), Legionella pneumophila (14), and higher plants (18, 23) use the second strategy, reducing Fe(III) to Fe(II) prior to uptake of the iron. The obligate pathogens Neisseria gonorrhoeae and Neisseria meningitidis use the third strategy, binding and deferrating human transferrin and lactoferrin (19); Hemophilus influenzae scavenges iron directly from heme and hemoglobin (24). Iron uptake mechanisms in pathogenic fungi are incompletely understood. Hydroxamate siderophores have been detected in culture supernatants of Histoplasma capsulatum (2). Rhizopus species express surface receptors for ferri-deferoxamine but do not secrete hydroxamate siderophores (1); thus, deferoxamine represents an iron uptake-related vitamin in Rhizopus, as it does in the pathogenic bacterium Yersinia enterocolitica (25). In Cryptococcus neoformans, deferoxamine stimulates growth in the presence of iron starvation, but this fungus does not secrete Fe(III) chelators, nor does it express a specific transferrin receptor (12, 13). Rather, C. neoformans and Candida albicans reduce extracellular Fe(III) to the much more soluble form, Fe(II), like S. cerevisiae, and are presumed to transport nascent Fe(II) into their cells (20, 22). In what has been termed nutritional immunity, vertebrates use unsaturated transferrins to withhold their iron from microbes and respond to infections by further sequestering their stores of iron, even to the point of anemia (3). This inference is supported by the observation that saturation with physiologic iron chelators leads to opportunistic infections (28). It has been hypothesized that the administration of therapeutic iron-sequestering agents may further protect host iron stores and thereby alleviate infections. Such compounds are being actively sought, but currently the only chelator which can be safely administered is deferoxamine, a microbial hydroxamate siderophore. One might expect that this compound would sequester iron and block iron uptake by microbes which lack receptors for the iron chelate; however, the compound also might be predicted to stimulate uptake in microbes which express such receptors. Indeed, it has been found that the administration of deferoxamine alleviates malaria (9) but promotes opportunistic infections by Rhizopus species (1). Thus, microbial iron uptake mechanisms can powerfully affect the host-parasite balance, and understanding them has the potential to lead to better management of infections. C. neoformans is the etiologic agent of a meningitis which complicates many immunodeficiency diseases, including 10 to 20% of those resulting from the human immunodeficiency virus (17). Although the symptoms of cryptococcosis can often be suppressed by treatment with antifungal agents, in most cases the infection is incurable and lifelong treatment is required (29). Because control of iron uptake has decided the outcome of infections in the model cases cited above, we investigated iron uptake in C. neoformans with the hope of being able to bolster nutritional immunity. We previously described two mechanisms by which this organism reduces extracellular iron: expression of a cell membrane ferric reductase and secretion of the nonspecific reductant 3-hydroxyanthranilic acid (22). In the present report, we describe iron uptake physiologically. We chose to use a serotype D strain because of the availability of both meiotic and molecular analyses in the serotype D cryptococcal system.