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Regulation of vacuolar trafficking by Vps45p in the pathogenic fungus Cryptococcus neoformans Nielson, Erik David


Virulence factor elaboration of the human pathogenic fungus Cryptococcus neoformans is strongly regulated by extracellular iron content and type. In addition, acquisition of iron from the host has been shown to be essential for pathogenic growth. Previously, the endosomal sorting complex required for transport was identified as an essential component of iron acquisition from the ferrophore heme. In this study, we further show that iron acquisition from hemin requires the translocation of this molecule to the vacuole by specific deletion of the gene encoding the vacuolar trafficking regulatory protein Vps45p. In addition to decreased ability to utilize extracellular hemin, this mutant is also retarded for growth in the presence of FeCl3, and is defective for cell wall integrity pathway responses when challenged by NaCl, sodium dodecyl sulphate, calcofluor white, or caffeine. These mutants have dramatically increased sensitivities to the vacuolar-accumulating drugs chloroquine and quinacrine. No associated defects in virulence factor elaboration such as loss of melanin deposition or extracellular capsule are observed. However, upon in vitro challenge by mouse J774a.1 and human THP-1 derived macrophage cell lines, mutants in the VPS45 gene were markedly unable to survive when compared to wild-type cells under identical conditions. These data underscore a growing theme in fungal genetics that the importance of vacuolar protein trafficking extends beyond nutrient storage. Vacuolar function in C. neoformans appears to strongly correlate with iron acquisition, extracellular signalling response with respect to cell wall integrity, and survival within the phagolysosomal compartment of macrophage-like cells

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