Objectives: Cryptococcus neoformans is the main causative agent of cryptococcal meningitis, a disease that is responsible for ~ 15% of AIDS-related deaths. In this regard, cryptococosis is the second most common cause of mortality in people with HIV/AIDS. Unfortunately, very few antifungal drugs are available to treat cryptococcal infections. However, understanding the mechanisms of C. neoformans pathogenesis may lead to new therapeutic approaches. The competition for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection, and C. neoformans employs multiple mechanisms to compete for iron during cryptococcosis. In this study, our goal was to examine the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. Methods: Cellular localizations were performed using fluorescence microscopy on Vps45-GFP and Cfo1-GFP strains stained with FM4-64 and MitoTracker Red CMXRos. Growth curves and growth assays on the WT strain, vps45 mutants and complemented strains were performed in the presence of trafficking inhibitors, different iron sources, calcium chelators, mitochondria electron transport chain inhibitors, inducers of reactive oxygen species (ROS), cell wall stressors, and antifungal and antimalarial drugs. Flow cytometry was used to assess ROS stress, mitochondrial membrane permeability and cell wall integrity. Virulence assays were performed in the murine macrophage-like cell line J774A.1 and in an inhalation mouse model of cryptococcosis. Results: As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein co-localized with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis.  Conclusion: These discoveries shed light on the molecular mechanisms underlying the uptake and use of iron as an essential nutrient for the virulence of C. neoformans. Further investigations could lead to the development of antifungal drugs that target Vps45-mediated processes.


The vacuolar sorting protein Vps45 links iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans.