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Heme utilization and storage by Cryptococcus neoformans. Scofield, Melanie


The opportunistic fungal pathogen Cryptococcus neoformans has been previously shown to use heme as a sole iron source, but the mechanisms for heme utilization are unknown. The goal of this study was to begin a genetic analysis of heme utilization in C. neoformans by deletion of candidate genes and phenotypic characterization. The first hypothesis was that a putative heme oxygenase protein, Hmx1, was responsible for degrading heme to release iron. However, an hmx1 deletion strain was capable of growth on heme, indicating that the gene is not required for heme utilization. The expression pattern of HMX1 showed down-regulation in the presence of heme and hemoglobin indicating that HMX1 likely plays a regulatory role within the cell. Because loss of the heme-related gene HMX1 did not reveal any phenotypes related to heme as an iron source, the role of the vacuolar protein Vps41 in iron and heme utilization was also examined. The work on Vps41 was designed to test a second more general hypothesis that the vacuole is involved in heme or iron storage and utilization. It was found that vps41 mutants had heme and iron growth defects, as well as increased sensitivity to excess levels of both heme and inorganic iron. Analysis of the wild-type strain grown with heme led to the surprising discovery of dark intracellular aggregates that were visible with light microscopy. These aggregates were reminiscent of the crystallized heme (hemozoin) found in malaria parasites. In contrast, the cells of vps41 mutants became filled with diffuse heme throughout the cell, indicating that an intact vacuole was required for aggregate formation. The inability of the mutant to sequester heme in the aggregates may contribute to the observed sensitivity of the strain to heme toxicity. Overall, these results provide new insights into heme utilization and storage in C. neoformans.

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