Date Published: August 2, 2018
Publisher: Public Library of Science
Author(s): Mélissa Caza, Guanggan Hu, Erik David Nielson, Minsu Cho, Won Hee Jung, James W. Kronstad, Damian J. Krysan.
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.
The pathogenic fungus Cryptococcus neoformans attacks immunocompromised people to cause cryptococcosis, a particularly devastating disease in HIV/AIDS sufferers . Adaptations of the fungus to cause disease in mammalian hosts include the ability to grow at 37°C, to deliver key virulence components to the external milieu, and to acquire nutrients for proliferation [2,3]. In the latter case, iron plays a key role in the virulence of C. neoformans as a cofactor in essential biochemical reactions and as a regulator of the elaboration of the polysaccharide capsule, a major virulence factor [4,5]. As with other pathogens, C. neoformans must compete against host nutritional immunity to obtain iron during infection. Iron withholding by the host is achieved by iron-binding proteins such as transferrin, lactoferrin, and ferritin that maintain available iron at extremely low levels [6,7]. On the other hand, iron overload exacerbates cryptococcal disease in a mouse model of cryptococcosis . 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  and the requirement of the endosomal sorting complex required for transport (ESCRT) pathway for endocytosis and intracellular trafficking of exogenous heme [12,13]. Furthermore, these systems are known to participate in the virulence of C. neoformans in a murine inhalation model of cryptococcosis [9–13].
The C. neoformans gene locus CNAG_03628.2 encoding a candidate Vps45 protein was first identified by a reciprocal BLASTp search using the amino acid sequence of S. cerevisiae Vps45. The predicted polypeptide (686 aa) from C. neoformans displayed 36% identity and 56% similarity to the S. cerevisiae protein and 47% identity and 63% similarity to the corresponding protein from Aspergillus nidulans. A phylogenetic analysis for Vps45 sequences is presented in Fig 1. Considering the strong relatedness between orthologs of VPS45, we proposed to change the annotation of CNAG_03628.2 to Vps45. To examine the role of Vps45 in C. neoformans, we constructed two independent targeted deletion mutants and corresponding strains in which the mutation was complemented with the wild-type (WT) gene. The genotypes of the strains were confirmed by PCR and genome hybridization (S1 Fig).
The SM protein Vps45 plays an important role in vesicle trafficking by conferring specificity on SNARE proteins that mediate docking and fusion events . As initially demonstrated in S. cerevisiae, a vps45 mutant is defective in vacuolar biogenesis due to impaired fusion of Golgi-derived vesicles with the prevacuolar compartment [28,46]. In this study, we investigated the role of an ortholog of Vps45 in iron trafficking in C. neoformans, and found that the protein is needed for robust growth of the pathogen on both inorganic and organic iron sources. Notably, the iron-related phenotypes were more severe at 37°C. Interestingly, loss of Vps45 changed the intracellular distribution of a fusion protein of GFP with the ferroxidase Cfo1 that mediates high affinity iron uptake in C. neoformans. Specifically, the WT strain accumulated Cfo1-GFP at the plasma membrane and vacuole under low iron conditions while the protein was found in the PM and internal punctate structures (presumably endosomes) in a vps45 mutant. That is, loss of Vps45 prevented vacuolar accumulation. Under high iron conditions, the protein accumulated in internal punctate structures in the WT strain, and in the PM and internal dispersed structures in the mutant. These results suggest that proper trafficking of Cfo1, including localization at the vacuole, contributes to robust growth on FeCl3. The differences in localization and growth were most marked at 37°C, and the mutant hyper-accumulated iron at this temperature. We speculate that proper trafficking of Cfo1 to the vacuole contributes to robust iron uptake, correct intracellular distribution, and overall homeostasis (Fig 10).