Abstract
The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined 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. 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 associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. 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 to 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. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.
Highlights
The pathogenic fungus Cryptococcus neoformans attacks immunocompromised people to cause cryptococcosis, a devastating disease in HIV/AIDS sufferers [1]
Because of the iron-limited nature in the host, C. neoformans has developed multiple strategies to acquire iron including the use of a high-affinity iron uptake system composed of the cell surface iron permease Cft1 and the ferroxidase Cfo1 [9,10], the secreted mannoprotein Cig1 for iron uptake from heme [11] and the requirement of the endosomal sorting complex required for transport (ESCRT) pathway for endocytosis and intracellular trafficking of exogenous heme [12,13]
Vps45 is required for iron transport and virulence in C. neoformans microscopy images were taken every 15 min for 90 min
Summary
The pathogenic fungus Cryptococcus neoformans attacks immunocompromised people to cause cryptococcosis, a devastating disease in HIV/AIDS sufferers [1]. Because of the iron-limited nature in the host, C. neoformans has developed multiple strategies to acquire iron including the use of a high-affinity iron uptake system composed of the cell surface iron permease Cft and the ferroxidase Cfo1 [9,10], the secreted mannoprotein Cig for iron uptake from heme [11] and the requirement of the endosomal sorting complex required for transport (ESCRT) pathway for endocytosis and intracellular trafficking of exogenous heme [12,13] These systems are known to participate in the virulence of C. neoformans in a murine inhalation model of cryptococcosis [9,10,11,12,13]
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