The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans

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.

Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for 23 vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a 24 vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking 25 inhibitors. The mutant also showed poor growth on iron-limited media and a defect in 26 transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma 27 membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also 28 contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with 29 mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron 30 transport complexes as well as changes in mitochondrial membrane potential. Consistent 31 with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To 32 assess the relevance of these defects for virulence, we examined cell surface properties of 33 the vps45 mutant and found increased sensitivity to agents that challenge cell wall The pathogenic fungus Cryptococcus neoformans attacks immunocompromised 55 people to cause cryptococcosis, a particularly devastating disease in HIV/AIDS sufferers 56 (1). Adaptations of the fungus to cause disease in mammalian hosts include the ability to 57 grow at 37 o C, to deliver key virulence components to the external milieu, and to acquire 58 nutrients for proliferation (2,3). In the latter case, iron plays a key role in the virulence 59 of C. neoformans as a cofactor in essential biochemical reactions and as a regulator of the 60 elaboration of the polysaccharide capsule, a major virulence factor (4,5). As with other 61 pathogens, C. neoformans must compete against host nutritional immunity to obtain iron 62 during infection. Iron withholding by the host is achieved by iron-binding proteins such 63 as transferrin, lactoferrin, and ferritin that maintain available iron at extremely low levels 64 (6,7). On the other hand, iron overload exacerbates cryptococcal disease in a mouse 65 model of cryptococcosis (8). Because of the iron-limited nature in the host, C. 66 neoformans has developed multiple strategies to acquire iron including the use of a high-67 affinity iron uptake system composed of the cell surface iron permease Cft1 and the 68 ferroxidase Cfo1 (9,10), the secreted mannoprotein Cig1 for iron uptake from heme (11) 69 and the requirement of the endosomal sorting complex required for transport (ESCRT) 70 pathway for endocytosis and intracellular trafficking of exogenous heme (12,13). 71 Furthermore, these systems are known to participate in the virulence of C. neoformans in 72 a murine inhalation model of cryptococcosis (9-13). 73 The discovery that the ESCRT pathway is critical for iron acquisition from Candida albicans. For example, the ferrichrome siderophore receptor Arn1p in S. 84 cerevisiae is found in endosome-like vesicles and is sorted directly from the Golgi to the 85 endosomal compartment in the absence of its substrate. However, when cells are exposed 86 to ferrichrome, Arn1p is relocalized to the plasma membrane where the siderophore-87 loaded receptor complex rapidly undergoes endocytosis (17). In C. albicans, the plasma 88 membranes proteins Rbt5 and Rbt51 are required for binding and iron uptake from heme 89 and hemoglobin. A screen for mutants defective in iron utilisation from hemoglobin in S. 90 cerevisiae expressing heterologously Rbt51 revealed mutants impaired in vacuolar 91 functions, including vacuolar ATPase, and components of the ESCRT pathway and the 92 HOPS complex. Subsequent analysis confirmed the role of ESCRT-I complex VPS23 and 93 VPS28 in iron uptake from hemoglobin, but not from ferrichrome, suggesting that the 94 processes of iron uptake from hemoglobin and ferrichrome are distinct (18). Likewise, 95 ESCRT mutants in C. neoformans show defects in iron acquisition from heme and 96 hemoglobin, but not from ferrichrome (12,13). In addition, there was an extended lag 97 phase for ESCRT mutants when grown in media containing iron chloride as a sole iron 98 source suggesting that ESCRT pathway plays a role in recycling the high-affinity iron 99 uptake system Cft1-Cfo1 to the plasma membrane (9,10). This idea is consistent with the 100 findings in S. cerevisiae that levels of iron influence cellular localization of the iron 101 permease/ferroxidase proteins Ftr1 and Fet3 (i.e., recycling back to the plasma membrane 102 in low levels of iron or targeting to the vacuole for degradation in high levels of iron); 103 these endocytic sorting processes also require the ESCRT pathway (19).

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Vesicle fusion events in endocytic compartments are a dynamic process that is 105 regulated by specific mediators such as tethering factors, SNAREs, Rab GTPases,  Vps45. To examine the role of Vps45 in C. neoformans, we constructed two independent 139 targeted deletion mutants and corresponding strains in which the mutation was 140 complemented with the wild-type (WT) gene. The genotypes of the strains were 141 confirmed by PCR and genome hybridization (S1 Fig.). Vps45 is required for membrane trafficking to the vacuole. 146 As mentioned above, endomembrane trafficking is conducted by specific 147 membrane fusion events that occur via the formation of SNARE bundles, with regulation 148 by SM proteins. To determine whether Vps45 plays a role in endocytosis in C. 149 neoformans, cells were grown in low iron conditions, transferred to high iron media,  Fig.). Moreover, endocytosis assays with FM4-64 were also 160 performed using the VPS45-GFP complemented mutant grown at 30°C and 37°C (S2B 161 Fig.). At 30°C, Vps45-GFP fusion protein was first localized at the plasma membrane at 162 0 min and then to the vacuolar membrane within 15 min (Fig. 2B). Vacuolar co-163 localization of Vps45-GFP was found after 30 min of incubation at 37°C (S2B Fig.). 164 Notably, introduction of the VPS45-GFP gene fusion into the vps45 mutant 165 complemented growth defects for a number of conditions at 30°C and 37°C (S3 Fig.).  Vps45 contributes to iron acquisition in a temperature dependent manner. 188 We have previously demonstrated that iron acquisition from exogenous heme  (Fig. 3B) . This result indicates that Vps45 is required for the use of FeCl 3 at 202 the host temperature (37°C), but not at lower temperature. We speculate that another 203 system may contribute to iron uptake at 30°C but may be insufficient at 37°C. Similar 204 results were obtained with spot assays on solid media (Fig. 3C). However, when 205 incubated with organic iron sources (i.e. heme, hemoglobin, transferrin and sheep blood), 206 the vps45 mutants exhibited a delay in growth at both 30˚C and 37˚C, as compared with 207 WT and complemented strains ( Fig. 3A and B) . Also, growth was inhibited for the vps45 208 mutants when incubated with hemoglobin and blood at 37˚C. Again, similar results were 209 obtained with spot assays on solid media (Fig. 3C). These results suggest that Vps45 is 210 required for the use of organic iron sources found in mammalian hosts. Interestingly, 211 when intracellular iron content was measured by inductively coupled plasma mass 212 spectrometry (ICP-MS) for cells grown in high iron conditions at 37˚C, the vps45 213 mutants displayed a greater amount of iron compared to the WT and complemented 214 strains (Fig. 3D). We hypothesize that the ability to properly traffic the components of 215 iron uptake systems may be required at 37°C. That is, loss of Vps45 may impede the 216 delivery of iron to the vacuole (and sensing of iron repletion) to create an imbalance in 217 iron homeostasis leading to enhanced iron uptake and the increase in intracellular iron 218 content in vps45 mutants. Altogether, the results indicate that Vps45 is required for 219 intracellular iron transport in a temperature-dependent manner. Vps45 contributes to endocytic transport of the ferroxidase Cfo1 for high-affinity 222 iron uptake. 223 In C. neoformans, iron from FeCl 3 or transferrin is transported by the high-affinity 224 iron uptake system composed of the iron permease Cft1 and the ferroxidase Cfo1 (9,10).

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Indeed, deletion of either CFT1 or CFO1 results in severe growth defects with FeCl 3 or 226 transferrin as sole iron sources, but not with heme or hemoglobin, which suggests that 227 there are distinct uptake pathways for these iron sources (9,10). The Cft1-Cfo1 system is  Fig.).

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Upon addition of FeCl 3 , the Cfo1-GFP signal was reduced in the WT and 248 complemented strains, and was found mostly in vesicular bodies after 24h of incubation 249 at 30˚C (Fig. 4B). The signal was also found in vesicular bodies in cells from the YNB 250 medium at 30˚C and, as mentioned above, the signal was mainly in the vacuole in cells 251 from YNB-BPS (iron-chelated) medium. With incubation at 37°C in YNB-BPS+FeCl 3 , a 252 strong signal was detected for Cfo1-GFP, and the fusion protein was found at the plasma 253 membrane, in punctate structures and at the vacuole in the WT and complemented strains 254 (S4B Fig.). In these strains, the signal was also found in the vacuole and vesicular bodies 255 in cells from the YNB medium at 37˚C, and the signal was mainly in the vacuole in cells 256 from YNB-BPS medium. Again, Cfo1-GFP was located mainly at the plasma membrane 257 in vps45 mutants when grown in YNB, YNB+BPS and YNB-BPS+FeCl 3 at 37°C for 24h 258 (S4B Fig.). We established that full length Cfo1-GFP protein was present in the WT, complemented strains (Fig. 5C). Increased sensitivity to hydrogen peroxide (H 2 O 2 ) was 292 also observed for the vps45 mutants, but no growth defect was noted when the mutants 293 were exposed to other agents that caused oxidative stress such as paraquat, plumbagin, 294 diphenyleneiodonium chloride (dpi) and menadione (Fig. 5C, and S5C Fig.).  and rod shaped mitochondria were observed when cells were grown in YNB at 30°C or 336 37°C, and no marked change in mitochondrial morphology was observed when cells were 337 exposed to the calcium chelator EGTA at either temperature.

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In light of our finding that Vps45 participates in mitochondrial functions, we next showed that Vps45-GFP protein was found associated with a subset of mitochondria 343 upon calcium restriction in 61% of cells grown at 30°C and 87% of cells grown at 37°C 344 ( Fig. 6E and F). We also measured the mitochondrial membrane potential (MMP) by 345 flow cytometry in cells stained with JC1-1 (S5D Fig.). When the WT and complemented 346 strains were grown under calcium-starved conditions (YNB-EGTA) at 30°C, a shift from 347 mixed polarized to depolarized mitochondria was observed. A similar shift was also 348 obtained for vps45 mutants but to a lesser extent. Interestingly an increased in 349 temperature restored mitochondrial membrane potential population to levels similar to 350 those found when cells were grown in YNB at 37°C (Fig. 6G). We went on to measure 351 intracellular calcium content by inductively coupled plasma mass spectrometry (ICP-352 MS), and found that only the vps45 mutants displayed a greater amount of calcium 353 compared to WT and complemented strains in high iron conditions at 37˚C (Fig. 6H).

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Taken together, these results suggest that Vps45 influences calcium homeostasis and 355 mitochondrial membrane potential in a manner related to iron availability. were sensitive to agents that challenge cell wall integrity (38,39). Specifically, we 366 exposed our strains to calcofluor white, SDS, caffeine or NaCl, and found that the vps45 367 mutants displayed growth defects, especially at 37˚C, when compared to the WT and 368 complemented strains (Fig.7A). Furthermore, deletion of CRZ1 in a vps45 mutant 369 abolished growth on calcofluor white and caffeine at 37°C (Fig. 7B). In addition, the and cell size increased when cells of the vps45 mutants were grown at 30˚C (Fig. 7D).

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However, a reduction in capsule size only was observed at 37˚C (Fig. 7F). 30°C, but not at 37°C (Fig. 8B). This result is in agreement with the previous results that 436 Vps45 is required for the use of FeCl 3 at the host temperature (37°C), but not at lower 437 temperature. Furthermore, addition of iron and heme to miconazole aided the growth of 438 WT and complemented strains, but not the vps45 mutants (Fig. 8B). Overall, these 439 results suggest that Vps45 is required for resistance to drugs that target the cell wall, 440 membranes and the vacuole. or any of the organs (i.e., lungs, brain, liver, spleen or kidney) (Fig. 9C). Overall, these

Construction of deletion mutants and complemented strains 581
All deletion mutants were constructed by homologous recombination using gene 582 specific knockout cassettes, which were amplified by three-step overlapping PCR (53) 583 with the primers listed in S1 Table. The resistance marker for nourseothricin (NAT) was 584 amplified by PCR using the primers Cassette F and Cassette R and the plasmid pCH233