Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus Neoformans

Journal of Fungi

Article Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

Donghyeun Kim 1 , Moonyong Song 1, Eunsoo Do 2, Yoojeong Choi 1, James W. Kronstad 3 and Won Hee Jung 1,*

1 Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea; [email protected] (D.K.); [email protected] (M.S.); [email protected] (Y.C.) 2 Department of Microbiology, University of Georgia, Athens, GA 30602, USA; [email protected] 3 Michael Smith Laboratories, Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; [email protected] * Correspondence: [email protected]; Tel.: +82-31-670-3068; Fax: +82-31-675-1381

Abstract: Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were

highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug

Citation: Kim, D.; Song, M.; Do, E.; ﬂuconazole caused increased vacuolar fragmentation. These conditions also commonly cause an Choi, Y.; Kronstad, J.W.; Jung, W.H. increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles Oxidative Stress Causes Vacuolar are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only Fragmentation in the Human Fungal localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly Pathogen Cryptococcus neoformans. J. due to copper or iron limitation. Fungi 2021, 7, 523. https://doi.org/ 10.3390/jof7070523 Keywords: Cryptococcus neoformans; fragmentation; oxidative stress; superoxide dismutase; vacuole

Academic Editor: Brian L. Wickes

Received: 19 May 2021 1. Introduction Accepted: 26 June 2021 Published: 29 June 2021 The vacuole is an important cellular organelle in fungi, and it performs diverse functions including the storage of ions, amino acids, phosphate, and carbohydrates. The

Publisher’s Note: MDPI stays neutral vacuole morphology also dynamically adapts to intra- and extracellular environmental with regard to jurisdictional claims in conditions through fusion (forming a single enlarged vacuole) and ﬁssion, which is also published maps and institutional afﬁl- called fragmentation. For example, vacuole fusion is induced by nutrient limitation or iations. hypotonic treatment, whereas vacuole fragmentation is triggered by hyperosmotic shock, endoplasmic reticulum stress, or lactic acid stress in the model fungus Saccharomyces cerevisiae [1,2]. The underlying mechanisms governing vacuole morphological changes are not fully understood. However, studies using S. cerevisiae suggest that increased vacuole volume by fusion enhances degradation of cellular materials through autophagy and mul- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. tivesicular body pathways [1]. Moreover, genetically increased vacuole fusion extends the This article is an open access article lifespan of S. cerevisiae [3]. Vacuole fragmentation is a complex process, and several proteins distributed under the terms and have been shown to be involved. Examples include Fab1, a lipid kinase that produces conditions of the Creative Commons phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] from phosphatidylinositol 3-phosphate Attribution (CC BY) license (https:// in S. cerevisiae. The S. cerevisiae mutant lacking the FAB1 gene exhibits an enlarged vacuole, creativecommons.org/licenses/by/ indicating the involvement of the protein in vacuole fragmentation [4]. In addition to 4.0/). Fab1, the target of rapamycin complex 1 (TORC1) is required for vacuole fragmentation

J. Fungi 2021, 7, 523. https://doi.org/10.3390/jof7070523 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, 523 2 of 13

in S. cerevisiae and cells treated with rapamycin do not display vacuole fragmentation [5]. The involvement of superoxide dismutase (SOD) in vacuole fragmentation has also been demonstrated [6]. Specifically, loss of S. cerevisiae Zn, Cu -superoxide dismutase Sod1, but not Mn-superoxide dismutase Sod2, led to significantly increased vacuole fragmentation [6]. SOD converts superoxide radicals into hydrogen peroxide and molecular oxygen. Therefore, increased vacuole fragmentation in the sod1 mutant implied an influence of superoxide radicals on vacuole morphology and function in S. cerevisiae. Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningitis in immunocompromised hosts. C. neoformans possesses two genes, SOD1 and SOD2, which are similar to the genes in S. cerevisiae and encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase, respectively. A number of biochemical and genetic studies have investigated the role of these enzymes in the physiology and virulence of C. neoformans [7–11]. For example, mutants lacking either SOD1 or SOD2 displayed increased susceptibility to oxidative stress and reduced growth at higher temperatures. Moreover, there was reduced survival of mice infected with the sod1 mutant, whereas disease was completely abolished with the sod2 mutant in a murine model of cryptococcosis, suggesting the importance of these enzymes in the pathogenesis of C. neoformans, although their contributions may be different [8,9]. Furthermore, a recent study revealed that the expression of SOD1 and SOD2 is directly regulated by Cuf1, the transcription factor that controls expression of the genes involved in copper homeostasis in C. neoformans. Indeed, the Cuf1 binding site (copper responsive elements) was identified within the promoter regions of both SOD1 and SOD2. The same study also identified two isoforms of Sod2, one is the canonical mitochondrial protein, and the other is a novel cytosolic isoform specifically expressed under copper-limiting conditions, thus revealing Cuf1 and copper-dependent regulation of Sod1 and Sod2 in C. neoformans [12]. In addition to the association of SODs with copper homeostasis, we recently showed the influence of SODs on iron homeostasis, and on the cytosolic heme pool in particular, in C. neoformans. By utilizing a cytosolic heme sensor, we demonstrated that deletion of SOD2 caused a significant reduction in intracellular heme levels due to increased ROS in the sod2 mutant, whereas the sod1 mutant displayed similar intracellular heme levels compared with the wild-type strain. In the same study, we also found that inhibition of vacuole function reduced intracellular heme levels, suggesting that the vacuole is an important organelle for maintaining heme homeostasis [13]. In this study, we addressed the role of SODs in C. neoformans in more detail, specifically focusing on iron homeostasis and vacuole morphology. The localization of both SODs was first confirmed by fusion with green fluorescent protein (GFP), and the influence of the enzymes on the major iron regulatory proteins, Cir1 and HapX, was investigated to understand the connection between the SOD proteins and iron homeostasis. We also studied the effect of deletion of SOD1 and SOD2 on vacuole morphology and the response to oxidative stress to determine the role of SODs in the physiology of C. neoformans. Finally, the mechanism by which Fab1 and TORC1 regulate vacuole morphology in C. neoformans was investigated.

2. Materials and Methods 2.1. Strains and Growth Media Cryptococcus neoformans var. grubii H99 was used as the wild-type strain. The sod1 and sod2 mutants used in the current study were constructed as previously described [13]. For fungal cultures, yeast extract-bacto peptone medium supplemented with 2% glucose (YPD) was used. To prepare media with low concentrations of iron, copper, or zinc, 100 µM of bathophenanthrolinedisulfonic acid (BPS), bathocuproinedisulfonic acid disodium salt, or N,N,N0,N0-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (Sigma, Munich, BY, Germany) were added to YPD, respectively. Media with high concentrations of iron, copper, or zinc were prepared by supplementing low-iron, low-copper, or low-zinc media with 250 µM FeCl3, CuSO4, or ZnCl2, respectively. J. Fungi 2021, 7, 523 3 of 13

2.2. Construction of Strains Expressing the Sod1-GFP or the Sod2-GFP Fusion Protein To construct the genes to express the Sod1-GFP or Sod2-GFP fusion proteins, the SOD1 and SOD2 genes were ampliﬁed by PCR using the wild-type genomic DNA and primers Sod1-GFP_F/Sod1-GFP_R, and Sod2-GFP_F/Sod2-GFP_R, respectively (Table S1). The PCR fragments for SOD1 and SOD2 were digested with HindIII or BamHI, respectively, and cloned into pWH091 as previously described [14]. The resulting plasmids were named pWH151 and pWH152 and were digested with XmaI and BspHI, respectively. Digested pWH151 and pWH152 plasmids were transformed to the sod1 or sod2 mutants, respectively, and positive transformants were selected and conﬁrmed by PCR and Western blot analysis.

2.3. Construction of the fab1 Knock-Out Mutant To construct the fab1 knock-out mutant, the genetic locus (CNAG_01209) containing the 8142-base pair open reading frame of the gene was replaced by homologous recombina- tion with a gene-specific deletion cassette, which was amplified using the genomic DNA of the wild-type strain and pCH233, which contains the nourseothricin acetyltransferase gene as a template. The amplified cassette was transformed into the wild-type strain by biolistic transformation [15]. Positive transformants were confirmed using PCR. Two independent mutants were analyzed and found to have the same phenotypes.

2.4. Confocal Fluorescence Microscopy To investigate the localization of Sod1 and Sod2, the strains expressing the Sod1-GFP or Sod2-GFP fusion proteins were grown in YPD medium at 30 ◦C overnight, diluted to an OD600 of 0.1 in fresh YPD medium, and incubated for 12 h. The cells were centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (PBS) and suspended in 50 µL of PBS or PBS containing MitoTracker (Invitrogen, Waltham, MA, USA). Cells were visualized using a confocal fluorescence microscope LSM800Airy (Carl Zeiss, Oberkochen, BW, Germany) at a magnifi- cation of 1000×; images were obtained using the ZEN 3.2 blue edition software (Carl Zeiss, Oberkochen, BW, Germany). To observe the vacuole, cells were grown in YPD medium overnight and diluted to an OD600 of 0.1 in fresh YPD medium or the same medium containing menadione, FeCl3, CuSO4, ZnCl2, or rapamycin as indicated in the figures. Cells were cultured to an OD600 of 1.0 and harvested by centrifugation at 5000 rpm for 5 min. The supernatant was discarded, and the cell pellet was suspended in 50 µL of YPD containing 0.5 µL of FM4-64 (a final concentration of 16 µM) (Thermo Fisher Scientific, Waltham, MA, USA). The cell suspension was incubated in the dark at 30◦C for 30 min, centrifuged at 5000 rpm for 3 min, washed twice with PBS, and suspended in 50 µL of PBS. The morphology and number of vacuoles in the cells were determined by confocal fluorescence microscopy as described above. To observe localization of the Sod2-GFP fusion protein in phagolysosomes, murine macrophage-like cells (J774A.1) were maintained in Dulbecco’s modified Eagle medium (DMEM; GenDEPOT, Katy, TX, USA) with 10% fetal bovine serum (Atlas, Fort Collins, CO, USA) and 100 µg/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37 ◦C in 5 5% CO2. Macrophages (5 × 10 cells) were seeded to a 24-well plate containing coverslips ◦ and incubated at 37 C in 5% CO2 overnight. C. neoformans cells were incubated in YPD at 30 ◦C overnight and opsonized with 9.34 µg/mL monoclonal 18B7 antibody at 37 ◦C for one hour. Macrophages were activated with 100 nM of phorbol 12-myritate 13-acetate ◦ (Sigma, St. Louis, MO, USA) at 37 C in 5% CO2 for one hour. Opsonized C. neoformans ◦ cells were coincubated with activated macrophages at 37 C in 5% CO2. After one hour, each well of the 24-well plate was washed three times with phosphate buffered saline (PBS) to remove non-phagocytized fungal cells, and the plate was incubated in fresh DMEM at 37 ◦C for 18 h. For fluorescence microscopic observation, a coverslip was removed from each well of the plate, washed five times with PBS, mounted on a glass slide using Prolong™ Gold Antifade Reagent (Invitrogen, Waltham, MA, USA), and localization of J. Fungi 2021, 7, 523 4 of 13

the Sod2-GFP protein in phagocytized cells was observed. In parallel, macrophages were also lysed by addition of sterilized deionized water, phagocytized C. neoformans cells were isolated, and localization of the Sod2-GFP was observed.

2.5. Flow Cytometric Analysis Cellular ROS levels were determined using MitoSOX-based assays and ﬂow cytometric ◦ analysis [16]. Cells were grown in YPD medium at 30 C overnight, diluted to an OD600 of 0.1 in fresh YPD medium and incubated at 30 ◦C for 12 h. The cells were collected, washed twice with fresh YPD medium, resuspended in YPD medium containing 5 µM MitoSOX (Thermo Fisher Scientiﬁc, Waltham, MA, USA), and incubated in the dark at 30 ◦C for 30 min. The cells were washed twice with PBS and suspended in 5 mL PBS. Flow cytometric analysis was performed using FACS AriaII (BD Bioscience, San Jose, CA, USA); data were analyzed using Flowing software (Turku Bioscience, Turku, Finland).

2.6. Western Blot Analysis ◦ Cells were grown in YPD medium at 30 C overnight and diluted to an OD600 of 0.1, in YPD medium containing 150 µM BPS to deplete iron. Cultured cells were harvested, diluted to an OD600 of 0.1 in YPD medium containing 150 µM BPS with or without 100 µM FeCl3, and incubated at 30 ◦C for 12 h. Cells were harvested by centrifugation and resuspended in a protein extraction buffer comprised of 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycolate, 1 mM PMSF, and a protease inhibitor cocktail (Sigma, Munich, BY, Germany). Cell lysates were prepared by bead-beating, and the protein concentration was determined by the Bradford assay [17]. Total proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma, Munich, BY, Germany). Protein detection was performed using an anti-GFP rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA) or an anti-FLAG (Abcam, Cambridge, UK) rabbit polyclonal antibody. A mouse anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz, Dallas, TX, USA) was used as a secondary antibody, followed by visualization by chemiluminescence.

2.7. Superoxide Dismutase Activity Assay Superoxide dismutase activity assay was performed as described previously [7]. Brieﬂy, cells were incubated in YPD containing 150 µM BPS with or without 250 µM FeCl3 at a concentration of 1 × 107 cells/mL for 12 h. The cells were harvested and lysed using native lysis buffer (50 mM Tris-Cl pH 7.5, 0.1 mM EDTA, 1 mM PMSF). The 20 µg of total protein was separated by native gel electrophoresis (132 mM Tris-base, 132 mM borate, 3.6 mM sodium citrate, 0.1% ammonium persulfate) and stained with nitro blue tetrazolium at room temperature for 30 min in dark. The gel was illuminated for 5 min and SOD activities in the wild-type strain, the sod1 mutant, and the sod2 mutant were visualized (Figure S1).

3. Results 3.1. Localization of Sod2 Is Influenced by Iron and Copper, and Iron Levels Impact the Expression and Activity of Sod2 We initially constructed strains expressing the Sod1-GFP or the Sod2-GFP fusion protein (Section2) to investigate localization of the proteins. The functionality of the fusion proteins was confirmed by growing the cells under high concentrations of NaCl and in the presence of a superoxide anion generating agent menadione, the conditions of which reduced the growth of the sod1 and sod2 mutants [7,18]. As shown in Figure1A, the strains expressing the Sod1-GFP or the Sod2-GFP fusion protein grew as well as the wild-type strain thus indicating the fusion proteins are fully functional. Examination of the intracellular localization of the fusion proteins by fluorescence microscopy revealed that Sod1-GFP and Sod2-GFP were localized in the cytoplasm and mitochondria, respectively, when the cells were grown in rich-medium (Figure1B). J. Fungi 2021, 7, 523 5 of 14

proteins was confirmed by growing the cells under high concentrations of NaCl and in the presence of a superoxide anion generating agent menadione, the conditions of which reduced the growth of the sod1 and sod2 mutants [7,18]. As shown in Figure 1A, the strains expressing the Sod1-GFP or the Sod2-GFP fusion protein grew as well as the wild-type strain thus indicating the fusion proteins are fully functional. Examination of the intracel- J. Fungi 2021, 7, 523 lular localization of the fusion proteins by fluorescence microscopy revealed that Sod1-5 of 13 GFP and Sod2-GFP were localized in the cytoplasm and mitochondria, respectively, when the cells were grown in rich-medium (Figure 1B).

Figure 1. Localization of SODs. ( (AA)) Functionality of of the the Sod1-GFP and and the Sod2-G Sod2-GFPFP fusion proteins was tested in spot assays of the strains on the medium containing NaCl or menadione. ( (BB)) Sod1 Sod1 and and Sod2 Sod2 were were each expressed as fusion proteins with GFP, and the respective cytosolic and mitochondrial localization of Sod1-GFP and Sod2-GFP was confirmed proteins with GFP, and the respective cytosolic and mitochondrial localization of Sod1-GFP and Sod2-GFP was conﬁrmed by fluorescent microscopy. FM4-64 and MitoTracker were used to visualize vacuoles and mitochondria, respectively. Im- by ﬂuorescent microscopy. FM4-64 and MitoTracker were used to visualize vacuoles and mitochondria, respectively. Images ages of two representative cells are presented for each strain. (C) Fungal cells grown in DMEM media showed mitochon- drialof two localization representative of the cells Sod2-GFP are presented protein for (DMEM) each strain. while ( Cthe) Fungal cells isolated cells grown from inthe DMEM phagol mediaysosome showed of the mitochondrialinfected mac- rophageslocalization displayed of the Sod2-GFP predominantly protein (DMEM)cytosolic whilelocalization the cells of isolated the protein from (Macrophage). the phagolysosome The ofcryptococcal the infected cells macrophages within a phagolysosomedisplayed predominantly of a macrophage cytosolic also localization showed cytosolic of the protein localization (Macrophage). of the Sod2-GFP The cryptococcal protein cells(Macrophage*). within a phagolysosome (D) Fluores- centof a macrophagemicroscopy of also Sod2-GFP showed localization cytosolic localization in cells grown of the in Sod2-GFPlow-copper protein (DMEM (Macrophage*). containing 50 (μDM) Fluorescentof bathocuproine microscopy disul- fonateof Sod2-GFP (BCS)) localizationor high-copper in cells (DMEM grown containing in low-copper 50 μM (DMEM BCS and containing 100 μM of 50 CuSOµM of4) bathocuproine medium, or low-iron disulfonate (DMEM (BCS)) con- or taining 50 μM of bathophenanthroline disulfonic acid (BPS)) or high-iron (DMEM containing 50 μM of BPS and 100 μM high-copper (DMEM containing 50 µM BCS and 100 µM of CuSO4) medium, or low-iron (DMEM containing 50 µM of of FeCl3) medium. bathophenanthroline disulfonic acid (BPS)) or high-iron (DMEM containing 50 µM of BPS and 100 µM of FeCl3) medium.

Recently,Recently, Smith Smith et et al. al. demonstrated demonstrated a cytoso a cytosoliclic isoform isoform of the of Sod2 the Sod2 protein protein upon upon cop- percopper limitation limitation [12]. [This12]. led This us ledto investigate us to investigate localization localization of Sod2-GFP of Sod2-GFP in the host in environ- the host mentenvironment of the phagolysosome of the phagolysosome of a murine of a murine macrophage-like macrophage-like cell line, cell where line, where the metal the metal con- centrationsconcentrations may may be significantly be significantly limited. limited. Cells Cells of the of C. the neoformansC. neoformans strainstrain expressing expressing the Sod2-GFPthe Sod2-GFP protein protein were were phagocytized phagocytized and and localization localization of ofthe the protein protein in in the the fungal fungal cells withinwithin phagolysosomes phagolysosomes was analyzed. Our Our results results indicated that Sod2-GFP localized not onlyonly in in mitochondria mitochondria but but also also in in the the cytosol cytosol in in internalized internalized C.C. neoformans neoformans cellscells (Figure(Figure 1C).1C). WeWe noted that not all phagocytized cells displayed cytosolic localization of the Sod2-GFP protein;protein; the the proportion proportion of of cells cells displaying displaying this this phenotype phenotype was was 67%. 67%. To confirm To confirm that cyto- that soliccytosolic localization localization of the of Sod2-GFP the Sod2-GFP fusion fusion protein protein was wasinfluenced influenced by copper by copper limitation, limitation, we investigatedwe investigated location location of the of protein the protein in cells in grown cells grown in medium in medium containing containing a copper a copper chelator.chelator. Indeed, Indeed, copper copper limitation limitation resulted resulted in partial in partial cytosolic cytosolic localization localization of of the the Sod2-GFP Sod2-GFP fusionfusion protein. protein. Furthermore, Furthermore, partial partial cytosolic cytosolic localization localization of of the the prot proteinein was was also also observed observed uponupon iron iron limitation limitation (Figure (Figure 11D).D). SuperoxideSuperoxide dismutase dismutase is is required required for maintaining intact iron-sulfur (Fe-S) clusters andand iron iron homeostasis, and itsits expressionexpression isis regulatedregulated by by intra- intra- and and extracellular extracellular iron iron levels. levels.For For example, example, cytosolic cytosolic Zn, Cu-superoxideZn, Cu-superoxid dismutasee dismutase SodA SodA expression expression is upregulated is upregulated upon iron depletion in both Aspergillus nidulans and A. fumigatus [19]. Additionally, increased expression of the cell wall-associated Cu-superoxide dismutase, Sod4, was also observed in C. albicans upon iron depletion [20]. In this context, we investigated whether the expression of the SOD proteins in C. neoformans was influenced by iron concentrations in the medium. Our results showed that the levels of Sod1 were generally higher than those of Sod2 and were independent of iron concentration in the medium. However, the level of Sod2 protein was significantly increased in the medium containing a high concentration of iron (Figure2A). These results suggest an association between Sod2 as a mitochondrial and J. Fungi 2021, 7, 523 6 of 14

upon iron depletion in both Aspergillus nidulans and A. fumigatus [19]. Additionally, increased expression of the cell wall-associated Cu-superoxide dismutase, Sod4, was also observed in C. albicans upon iron depletion [20]. In this context, we investigated whether the expression of the SOD proteins in C. neoformans was influenced by iron concentrations J. Fungi 2021, 7, 523 in the medium. Our results showed that the levels of Sod1 were generally higher6 than of 13 those of Sod2 and were independent of iron concentration in the medium. However, the level of Sod2 protein was significantly increased in the medium containing a high concentration of iron (Figure 2A). These results suggest an association between Sod2 as a mito- cytosolicchondrial Mn-SOD, and cytosolic and ironMn-SOD, homeostasis and iron in homeostasisC. neoformans in. C. However, neoformans our. However, results were our differentresults were from different those observed from those with observedAspergillus withand AspergillusCandida ,and suggesting Candida, thatsuggesting Sod2 is that the majorSod2 SODis the andmajor is significantlySOD and is significantly influenced influenced by iron levels by iron in C. levels neoformans in C. neoformans. Moreover,. More- our conclusionover, our wasconclusion further was supported further by supported the observation by the ofobservation significantly of increasedsignificantly Sod2 increased activity inSod2 wild-type activity or insod1 wild-typemutant or cells sod1 grownmutant in cells the grown presence in the of presence a high concentration of a high concentra- of iron (Figuretion of2 B).iron (Figure 2B).

FigureFigure 2. 2.Protein Protein levels levels and and activity activity of of Sod2 Sod2 are are influenced influenced byby iron,iron, andand deletiondeletion ofof SOD2SOD2 causedcaused reducedreduced Cir1 Cir1 and and HapX HapX levels. levels. ( A(A)) Strains Strains expressing expressing Sod1-GFPSod1-GFP oror Sod2-GFPSod2-GFP werewere growngrown in media withwith (+) (+) or or without without (− ()− 100) 100µM μM FeCl FeCl3, and3, and Western Western blot blot analysis analysis was was performed performed using using total total cell lysates.cell ly- ′ Thesates. membrane The membrane was stained was stained with copper with copper phthalocyanine-3,4 phthalocyanine-3,40,400,4000,4-tetrasulfonic″,4‴-tetrasulfonic acid tetrasodiumacid tetraso- (CPTA)dium (CPTA) to confirm to confirm equal loading equal ofloading each sample. of each (sample.B) The Sod2(B) The activities Sod2 activities of the strains of the grown strains in grown media in media with (+) or without (−) 100 μM FeCl3 were measured by native gel electrophoresis and with (+) or without (−) 100 µM FeCl were measured by native gel electrophoresis and nitroblue nitroblue tetrazolium staining. (C) The3 growth of the wild-type and the sod2 mutant were monitored tetrazoliumin YPD medium. staining. (D) ( CCellular) The growth ROS levels of the in wild-type the strains and were the determinedsod2 mutant using were the monitored MitoSOX-based in YPD medium.assays. ROS (D) Cellularlevels of ROSthe sod1 levels and in sod2 the strainsmutants were were determined compared with using those the MitoSOX-basedof the wild-type assays.strain. ROSAverages levels offrom the threesod1 and independentsod2 mutants experiments were compared are presented with those with of standard the wild-type deviations strain. (* Averagesp ≤ 0.05). from(E). threeStrains independent expressing Cir1-FLAG experiments in are the presented wild-typewith or the standard sod2 mutant deviations were grown (* p ≤ in0.05). media (E) with Strains (+) expressingor without Cir1-FLAG (−) 100 μM in FeCl the3 wild-type; Western orblot the analysissod2 mutant was perfor weremed grown and in the media membrane with (+) was or withoutstained with CPTA. (F) Strains expressing HapX-FLAG in the wild-type or the sod2 mutant were grown in (−) 100 µM FeCl3; Western blot analysis was performed and the membrane was stained with CPTA. media with (+) or without (−) 100 μM FeCl3; Western blot analysis was performed and the membrane (F) Strains expressing HapX-FLAG in the wild-type or the sod2 mutant were grown in media with (+) was stained with CPTA. or without (−) 100 µM FeCl3; Western blot analysis was performed and the membrane was stained with3.2. CPTA. Deletion of SOD Influences Expression of Iron Regulators as Well as Vacuole Morphology 3.2. DeletionWe next of SOD characterized Influences Expressionthe phenotypes of Iron of Regulators mutants as lacking Well as SOD1 Vacuole or Morphology SOD2 (con- structedWe next in our characterized recent study) the [13]. phenotypes Growth ofanalysis mutants showed lacking thatSOD1 the mutantor SOD2 lacking(constructed SOD1 ingrew our recentlike the study) wild-type [13]. strain Growth in YPD analysis medi showedum (data that not theshown), mutant but lacking the mutantSOD1 lackinggrew Δ likeSOD2 the displayed wild-type a strain reduced in YPD doubling medium time (data (Figure not 2C) shown), (WT: but84.6 themin; mutant sod2 : lacking93.6 min).SOD2 We displayedreasoned athat reduced the slower doubling growth time of the (Figure sod22 mutantC) (WT: may 84.6 be min; causedsod2 mainly∆: 93.6 by min). failure We to reasoned that the slower growth of the sod2 mutant may be caused mainly by failure to handle oxidative stress, and therefore we measured ROS accumulation in the cells. We found that compared with the wild-type strain, the sod2 mutant displayed significantly increased intracellular ROS levels. The sod1 mutant also showed increased ROS levels, but to a lesser extent than the sod2 mutant (Figure2D). Increased expression and activity of Sod2 in the wild-type cells at an elevated iron concentration and elevated ROS levels in the mutant implied that the sod2 mutant is deficient in iron homeostasis. Therefore, we investigated the expression of Cir1 and HapX, J. Fungi 2021, 7, 523 7 of 14

handle oxidative stress, and therefore we measured ROS accumulation in the cells. We found that compared with the wild-type strain, the sod2 mutant displayed significantly J. Fungi 2021, 7, 523 increased intracellular ROS levels. The sod1 mutant also showed increased ROS levels,7 of but 13 to a lesser extent than the sod2 mutant (Figure 2D). Increased expression and activity of Sod2 in the wild-type cells at an elevated iron con- thecentration major ironand transcriptionelevated ROS factorslevels in in theC. neoformansmutant implied, in the thatsod2 themutant. sod2 mutant Previously, is deficient it was in showniron homeostasis. that ironincreased Therefore, the we stabilityinvestigated and th abundancee expression of of Cir1 Cir1 in and wild-type HapX, the cells major [21]. iron In thetranscription current study, factors we in confirmed C. neoformans increased, in the sod2 Cir1 mutant. protein Previously, levels in wild-type it was shown cells growingthat iron underincreased high-iron the stability conditions. and abundance However, of Cir1 levels in wild-type of Cir1 were cells significantly [21]. In the current reduced study, inthe we sod2confirmedmutant, increased especially Cir1 protein in the highlevels iron in wild-type condition, cells indicating growing under that Sod2high-iron influences conditions. the abundanceHowever, levels of Cir1 of Cir1 (Figure were2E). significantly The influence reduced on HapX in the protein sod2 mutant, levels especially was more in dramatic the high becauseiron condition, deletion indicating of SOD2 thatreduced Sod2 the influence abundances the ofabundance the protein of inCir1 both (Figure high- 2E). and The low-iron influ- conditionsence on HapX (Figure protein2F). levels These was results more suggest dramatic that because Sod2 is criticaldeletion for of maintainingSOD2 reduced proper the abun- iron regulationdance of the mediated protein in by both the majorhigh- and iron low-iron regulators, conditions Cir1 and (Figure HapX, 2F). although These theresults underlying suggest mechanism(s)that Sod2 is critical require for maintaining further investigation. proper iron regulation mediated by the major iron regulators, VacuoleCir1 and fragmentationHapX, although was the oneunderlying of the key mechanism(s) phenotypes require observed further in S. investigation. cerevisiae SOD mutants,Vacuole particularly fragmentation for the wassod1 onemutant of the [6 key]. Accordingly, phenotypes weobserved investigated in S. cerevisiae the morphology SOD mu- oftants, the particularly vacuoles in thefor theC. neoformans sod1 mutantmutants [6]. Accordingly, lacking SOD1 we inveor SOD2stigatedby the confocal morphology fluorescence of the microscopy.vacuoles in the While C. neoformans the majority mutants of the lacking wild-type SOD1 cells or SOD2 showed by aconfocal single vacuole,fluorescence an average micros- ofcopy. two While vacuoles the majority per cell of was the observed wild-type in cells the showedsod1 mutant. a single In vacuole, contrast, an theaverage number of two of vacuolesvacuoles wasper cell dramatically was observed increased in the insod1 the mutant.sod2 mutant, In contrast, with anthe average number of of four vacuoles vacuoles was perdramatically cell, resembling increased vacuole in the fragmentationsod2 mutant, with in S. an cerevisiae average(Figure of four3 ).vacuoles These results per cell, suggest resem- thatbling activity vacuole of fragmentation both SODs is instrumentalin S. cerevisiae in (Figure preventing 3). These vacuolar results fragmentation, suggest that activity but Sod2 of playsboth SODs a more is instrumental important role in preventing in maintaining vacuolar the fragmentation, proper morphology but Sod2 of plays the vacuolea more im- in C.portant neoformans role in. maintaining the proper morphology of the vacuole in C. neoformans.

FigureFigure 3.3. DeletionDeletion ofof SOD1SOD1 oror SOD2SOD2 causedcaused vacuolevacuole fragmentation.fragmentation. ((AA)) CellsCells werewere growngrown inin YPDYPD medium,medium, stainedstained withwith FM4-64,FM4-64, andand theirtheir vacuole vacuole morphology morphology was was analyzed analyzed by by confocal confocal ﬂuorescence fluorescence microscopy. microscopy. (B) ( TheB) The number number of vacuoles of vacuoles per cellper ofcell each of each strain strain (the vertical(the vertical axis) axis) was quantiﬁed.was quantified. Averages Averages from from 50 independent 50 independent cells ofcells each of straineach strain are presented are presented with standardwith standard deviations deviations (* p ≤ (*0.05; p ≤ 0.05; ** p ≤ **0.001). p ≤ 0.001).

3.3. Oxidative Stress Induces Vacuole FragmentationFragmentation One of thethe keykey rolesroles ofof SODsSODs inin cellscells isis toto neutralizeneutralize ROS,ROS, andand mutantsmutants lackinglacking SODSOD thereforetherefore normallynormally showshow increasedincreased susceptibilitysusceptibility toto variousvarious oxidativeoxidative stresses.stresses. Sod2Sod2 hashas previouslypreviously beenbeen shownshown toto mediate protection against oxidative stress in C. neoformans [12].[12]. Here, we observed that that deletion deletion of of SOD1SOD1 oror SOD2SOD2 (to(to a greater a greater extent) extent) increased increased intracel- intra- cellularlular ROS ROS levels levels and and caused caused increased increased susceptibility susceptibility to oxidative to oxidative stress. stress. Therefore, Therefore, we rea- we reasonedsoned that that vacuole vacuole fragmentation fragmentation would would also be also induced be induced by increased by increased oxidative oxidative stress. To stress. test Tothis test hypothesis, this hypothesis, we induced we induced oxidative oxidative stress in stress wild-type in wild-type cells by cellsgrowth by in growth medium in medium contain- containinging menadione menadione and subsequently and subsequently analyzed analyzed the vacuole the vacuolemorphology morphology of the cells. of theThis cells. ap- Thisproach approach revealed revealed significantly signiﬁcantly increased increased vacuole fragmentation vacuole fragmentation in cells treated in cells with treated menadione with menadioneconfirming that conﬁrming oxidative that stress oxidative induced stress vacuole induced fragmentation vacuole fragmentation in C. neoformans in C. (Figure neoformans 4). (Figure4).

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J. Fungi 2021, 7, 523 8 of 13 J. Fungi 2021, 7, 523 8 of 14

Figure 4. Oxidative stress increased vacuole fragmentation. (A) Wild-type cells were grown in YPD medium with or without menadione (0.1 μg/mL), stained with FM4-64, and their vacuole morphology was analyzed by confocal fluorescence microscopy. (B) The number of the vacuoles per cell of each strain (the vertical axis) was quantified. Averages from 50 independent cells of each strain are presented with standard deviations (** p ≤ 0.001).

As mentioned above, we observed that an elevated concentration of iron increased the expression of Sod2, which suggested that this metal causes an oxidative stress response and increases Sod2 protein expression. To establish whether excess iron causes vacuole fragmentation, wild-type cells were grown in a medium containing high concentrations of iron, and their vacuoles were observed. Copper and zinc were also tested. The results showed that high concentrations of each of the metals increased vacuole fragmentation Figure(FigureFigure 4.4. 5A). OxidativeOxidative Moreover, stress stress our increased increased results vacuoleindicated vacuole fragmentation. fragmentation. that a main contributor(A) ( AWild-type) Wild-type to cells metal-mediated cells were were grown grown in vacu- YPD in YPDolemedium fragmentation medium with withor without was or without the menadione increase menadione (0.1in ROS μg/mL), (0.1 levelsµg/mL), stained in the stainedwith cells FM4-64, treated with FM4-64, andwith their high and vacuole concentrations their morphol- vacuole morphologyofogy the was metals analyzed was (Figure analyzed by confocal5B). by confocalfluorescence ﬂuorescence microscopy. microscopy. (B) The number (B) The of number the vacuoles of the per vacuoles cell of pereach cell Growingstrain of each (the strainC. vertical neoformans (the axis) vertical wa cellss axis)quantified. at 37 was °C—the quantiﬁed. Averages host Averages frombody 50 temperature—is independent from 50 independent cells known of each cells to strain ofinduce each are presented with standard deviations (** p ≤ 0.001). strainoxidative are presented stress [22]. with Additionally, standard deviations the sod2 (**mutantp ≤ 0.001). also displays a severe growth defect at 37 °C, presumably due to increased susceptibility of the cells to ROS generated at this tempera- As mentioned above, we observed that an elevated concentration of iron increased ture As[9]. mentionedC. neoformans above, cells we also observed suffer from that anoxidative elevated stress concentration when they of are iron treated increased with the an the expression of Sod2, which suggested that this metal causes an oxidative stress re- expressionazole antifungal of Sod2, drug, which such suggested as fluconazole, thatthis which metal not causesonly inhibits an oxidative ergosterol stress biosynthesis response and but sponse and increases Sod2 protein expression. To establish whether excess iron causes increasesalso increases Sod2 the protein production expression. of ROS To in establishthe fungus whether [23,24]. excessTherefore, iron we causes hypothesized vacuole frag- that vacuole fragmentation, wild-type cells were grown in a medium containing high concen- mentation,growing wild-type wild-type cells cells at were37 °C grown or treating in a medium them with containing fluconazole high would concentrations cause oxidative of iron, trations of iron, and their vacuoles were observed. Copper and zinc were also tested. The andstress their and, vacuoles in turn, increase were observed. vacuole Copperfragmentation. and zinc To were test this also idea, tested. weThe cultured results the showed cells at results showed that high concentrations of each of the metals increased vacuole fragmentation that37 °C high or in concentrations the presence of of fluconazole each of the and metals found increased that both vacuole conditions fragmentation increased vacuole(Figure 5frag-A) . (Figure 5A). Moreover, our results indicated that a main contributor to metal-mediated vacu- Moreover,mentation (Figure our results 5C). indicatedFurthermore, that significantly a main contributor increased to intracellular metal-mediated ROS levels vacuole were frag- ob- ole fragmentation was the increase in ROS levels in the cells treated with high concentrations mentationserved in the was cells the grown increase under in ROS these levels conditions, in the cellsa finding treated consistent with high with concentrations oxidative stress of of the metals (Figure 5B). thecausing metals vacuole (Figure fragmentation5B). (Figure 5D). Growing C. neoformans cells at 37 °C—the host body temperature—is known to induce oxidative stress [22]. Additionally, the sod2 mutant also displays a severe growth defect at 37 °C, presumably due to increased susceptibility of the cells to ROS generated at this temperature [9]. C. neoformans cells also suffer from oxidative stress when they are treated with an azole antifungal drug, such as fluconazole, which not only inhibits ergosterol biosynthesis but also increases the production of ROS in the fungus [23,24]. Therefore, we hypothesized that growing wild-type cells at 37 °C or treating them with fluconazole would cause oxidative stress and, in turn, increase vacuole fragmentation. To test this idea, we cultured the cells at 37 °C or in the presence of fluconazole and found that both conditions increased vacuole fragmentation (Figure 5C). Furthermore, significantly increased intracellular ROS levels were observed in the cells grown under these conditions, a finding consistent with oxidative stress causing vacuole fragmentation (Figure 5D).

FigureFigure 5.5. HighHigh concentrationconcentration ofof iron,iron, copper,copper, oror zinc,zinc, andand highhigh temperaturetemperature oror fluconazolefluconazole treatment,treatment, inducedinduced vacuolevacuole fragmentation. (A) Wild-type cells were grown in media with low or high levels of metals (250 μM FeCl3, CuSO4, or ZnCl2). fragmentation. (A) Wild-type cells were grown in media with low or high levels of metals (250 µM FeCl3, CuSO4, or ZnCl2). CellsCells werewere stainedstained withwith FM4-64,FM4-64, andand theirtheir vacuolevacuole morphologymorphology waswas analyzedanalyzed byby confocal fluorescencefluorescence microscopy.microscopy. ThreeThree independent cells from three independent experiments are presented. The bar graph presents the number of vacuoles per independent cells from three independent experiments are presented. The bar graph presents the number of vacuoles cell (the vertical axis) in each condition based on the averages from 50 independent cells of each strain with standard per cell (the vertical axis) in each condition based on the averages from 50 independent cells of each strain with standard deviations (** p ≤ 0.001). (B) Cellular ROS levels were determined using MitoSOX-based assays. Averages from three independent experiments are presented with standard deviations (* p ≤ 0.05; ** p ≤ 0.001). (C) Wild-type cells were grown at 37 ◦C or in medium containing fluconazole (FLZ) as indicated. Cells were stained with FM4-64, and their vacuole morphology was analyzed using confocal fluorescence microscopy. Three independent cells from three independent experiments are presented. The bar graph presents the number of vacuoles per cell (the vertical axis) in each condition based on the averages from 50 independent cells of each strain with standard deviations (** p ≤ 0.001). (D) Cellular ROS levels in the strains grown at 37 ◦C or in medium containing fluconazole were determined using MitoSOX-based assays. Figure 5. High concentration of iron, copper, or zinc, and high temperature or fluconazole treatment, induced vacuole The average of the values from three independent experiments with standard deviations are presented (* p ≤ 0.05). fragmentation. (A) Wild-type cells were grown in media with low or high levels of metals (250 μM FeCl3, CuSO4, or ZnCl2). Cells were stained with FM4-64, and their vacuole morphology was analyzed by confocal fluorescence microscopy. Three independent cells from three independent experiments are presented. The bar graph presents the number of vacuoles per cell (the vertical axis) in each condition based on the averages from 50 independent cells of each strain with standard

J. Fungi 2021, 7, 523 9 of 13

Growing C. neoformans cells at 37 ◦C—the host body temperature—is known to induce J. Fungi 2021, 7, 523 9 of 14 oxidative stress [22]. Additionally, the sod2 mutant also displays a severe growth defect at 37 ◦C, presumably due to increased susceptibility of the cells to ROS generated at this temperature [9]. C. neoformans cells also suffer from oxidative stress when they are treated deviations (** p ≤ 0.001). (B)with Cellular an azoleROS levels antifungal were determined drug, such using as fluconazole,MitoSOX-based which assays. not Averages only inhibits from three ergosterol independent experiments are biosynthesispresented with but standard also increases deviations the (* p production≤ 0.05; ** p ≤ 0.001). of ROS (C) in Wild-type the fungus cells [ 23were,24 grown]. Therefore, at 37 °C or in medium containingwe hypothesized fluconazole (FLZ) that as growing indicated. wild-type Cells were cells stained at 37 with◦C orFM4-64, treating and them their withvacuole fluconazole morphology was analyzed usingwould confocal cause fluorescence oxidative microscopy stress and,. Three in turn, independent increase cells vacuole from three fragmentation. independent Toexperi- test this ments are presented. The baridea, graph we presents cultured the number the cells of atvacuoles 37 ◦C per or cell in the(the presencevertical axis) of in fluconazole each condition and based found on that the averages from 50 independentboth conditions cells of each increased strain with vacuole standard fragmentation deviations (** (Figure p ≤ 0.001).5C). (D Furthermore,) Cellular ROS significantly levels in the strains grown at 37 °C orincreased in medium intracellular containing flucon ROS levelsazole were were determined observed inusing the MitoSOX-based cells grown under assays. these The conditions, average of the values from three independent experiments with standard deviations are presented (* p ≤ 0.05). a finding consistent with oxidative stress causing vacuole fragmentation (Figure5D).

3.4. Vacuole Fragmentation Is Also InfluencedInfluenced by Fab1 and TORC1 As mentionedmentioned previously,previously, Fab1 Fab1 is is involved involved in in vacuole vacuole fragmentation fragmentation in S.in cerevisiaeS. cerevisiae[4]. Therefore,[4]. Therefore, we investigatedwe investigated the influencethe influenc ofe the of Fab1the Fab1 homolog homolog on Sod2-mediatedon Sod2-mediated vacuole vac- fragmentationuole fragmentation in C. in neoformans C. neoformans. Specifically,. Specifically, we generated we generated a fab1 amutant fab1 mutant andinvestigated and investi- vacuolegated vacuole morphology morphology (Figure (Figure6). The 6).fab1 Themutant fab1 mutant possessed possessed a single a but single enlarged but enlarged vacuole pervacuole cell uponper cell growth upon in growth YPD medium, in YPD andmedium, its vacuole and its morphology vacuole morphology was unaffected was by unaf- the additionfected by of the metals, addition in contrast of metals, to thein contrast observations to the for observations wild-type cells. for wild-type These results cells. suggest These thatresults Fab1 suggest is an upstream that Fab1 componentis an upstream that component influences vacuole that influences fragmentation vacuole in fragmentationC. neoformans (Figurein C. neoformans6). In addition (Figure to Fab1,6). In TORC1addition is to also Fab1, required TORC1 for is vacuole also required fragmentation for vacuole in S. frag- cere- visiaementation[5,25 ].in Therefore, S. cerevisiae we. [5,25]. tested Therefore, whether TORC1we tested also whether influences TORC1 vacuole also influences fragmentation vac- inuoleC. fragmentation neoformans by growingin C. neoformans wild-type by andgrowingsod2 mutantwild-type cells and with sod2 rapamycin. mutant cells Wild-type with ra- S.pamycin. cerevisiae Wild-typecells were S. included cerevisiae as cells controls. were Theincluded results asshowed controls. that, The similar results to showedS. cerevisiae that, cells,similar vacuole to S. cerevisiae fragmentation cells, vacuole was significantly fragmentation inhibited was bysignificantly rapamycin inhibited in both the by wild-type rapamy- andcin in the bothsod2 themutant wild-type cells and in either the sod2 YPD mutant or high-iron cells in medium either YPD (Figure or high-iron7). This suggests medium that,(Figure similar 7). This to S.suggests cerevisiae that,, the similar TOR signalingto S. cerevisiae pathway, the TOR regulates signaling vacuole pathway fragmentation regulates invacuoleC. neoformans fragmentation, likely in with C. Fab1.neoformans Although, likely we with observed Fab1. Although the involvement we observed of Fab1 the and in- TORC1volvement in vacuoleof Fab1 fragmentationand TORC1 in invacuoleC. neoformans fragmentation, the mechanism in C. neoformans by which, the SODsmechanism affect theby which function SODs of these affect proteins the function still needs of these to beproteins explored. still needs to be explored.

FigureFigure 6.6. Fab1Fab1 inﬂuencesinfluences vacuolevacuole fragmentation.fragmentation. ( A) Wild-type and fab1fab1 mutant cells werewere growngrown inin high-metalhigh-metal mediamedia containingcontaining 250250µ μMM of of FeCl FeCl3,3 CuSO, CuSO4,4 or, or ZnCl ZnCl2,2 stained, stained with with FM4-64, FM4-64, and and their their vacuole vacuole morphology morphology was was analyzed analyzed by confocal by con- ﬂuorescencefocal fluorescence microscopy. microscopy. Three Three independent independent cellsfrom cells threefrom three independent independent experiments experiments are presented. are presented. (B) The (B) number The num- of vacuolesber of vacuoles in a cell in grown a cell grown in each in condition each condition was counted was counted (the vertical (the vertical axis), and axis), averages and averages from 50 from independent 50 independent cells of eachcells strainof each are strain presented are presented with standard with standard deviations deviations (** p ≤ 0.001). (** p ≤ 0.001).

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Figure 7.7. VacuoleVacuole fragmentation fragmentation is inhibitedis inhibited by rapamycin.by rapamycin. (A) Cells(A) Cells were were grown grown in YPD in or YPD high-iron or high-iron media withmedia or withoutwith or rapamycin,without rapamycin, stained withstained FM4-64, with FM4-64, and their and vacuole their morphologyvacuole morphology was analyzed was analyzed using confocal using confocal ﬂuorescence fluorescence microscopy. mi- Threecroscopy. independent Three independent cells from threecells from independent three independent experiments experiments are presented. are (presented.B,C). The number(B,C). The of vacuolesnumber of in vacuoles a cell grown in a incell each grown condition in each was condition counted, was and counted, averages and from averages 50 independent from 50 independent cells of each straincells of are each presented strain are with presented standard with deviations standard deviations (* p ≤ 0.05; ** p ≤ 0.001). S. cerevisiae BY4741 was included as a control. The vertical axis represents the (* p ≤ 0.05; ** p ≤ 0.001). S. cerevisiae BY4741 was included as a control. The vertical axis represents the number of vacuoles. number of vacuoles. Results present the average of the values from three independent experiments. Results present the average of the values from three independent experiments.

4. Discussion Trace metals such as iron, zinc, and copper are essential nutrients for every every organism. organism. However, excess amounts of these metals generate ROS and have a detrimentaldetrimental effect onon the cell. cell. In In the the current current study, study, we we found found that that high high concentrations concentrations of iron of iron as well as well as zinc as zincand andcopper copper induced induced vacuole vacuole fragmentation fragmentation in inwild-type wild-type C.C. neoformans neoformans cellscells likely likely because these metals increased intracellular ROS accumulation.accumulation. Iron overload is knownknown toto causecause the production of reactive free radicals viavia the Fenton reaction [[26–28].26–28]. Therefore, the high concentration of iron in the medium may have increased the intracellular labile iron pool, which in turn generated ROS. Excess zinc can also cause an increase in ROS accumulation, accumulation, and disrupt iron metabolism, such as Fe-S cluster synthesis,synthesis, inin S.S. cerevisiaecerevisiae [[29,30].29,30]. ExcessExcess copper also also generates generates ROS ROS and and causes causes oxidative oxidative stress, stress, DNA DNA damage, damage, and and membrane membrane dis- disruptionruption in S. in cerevisiaeS. cerevisiae [31,32].[31, 32Furthermore,]. Furthermore, similar similar to the to excess the excess amount amount of zinc, of Fe-S zinc, clus- Fe- Ster cluster synthesis synthesis is inhibited is inhibited by copper by copper toxicity toxicity [33]. Overall, [33]. Overall, cells possess cells possessmultiple multiple mecha- mechanismsnisms to maintain to maintain intracellular intracellular metal metalhomeostasi homeostasiss that can that be can disrupted be disrupted by elevated by elevated ROS. ROS.In C. neoformans, iron homeostasis has garnered much attention because of its in- volvementIn C. neoformans in the expression, iron homeostasis of virulence has garnered factors muchsuch as attention the polysaccharide because of its involve-capsule ment[34,35]. in Cir1 the expression and HapX ofare virulence two well-characterized factors such as theiron polysaccharide regulatory transcription capsule [34 factors,35]. Cir1 in andC. neoformans HapX are. The two protein well-characterized abundance of iron Cir1regulatory and HapX are transcription both influenced factors by in ironC. neofor-levels. mansUnder. The high-iron protein conditions, abundance Cir1 of Cir1 is more and abundant, HapX are bothand the influenced protein acts by iron as a levels. repressor Under for high-irongenes, including conditions, those Cir1 involved is more in abundant,iron uptake and and the metabolism. protein acts In as contrast, a repressor HapX for mainly genes, includingacts as a repressor those involved under low-iron in iron uptake conditions and metabolism.to inhibit the In expression contrast, HapXof genes mainly involved acts asin iron-consuming a repressor under functions low-iron [36] conditions. In the current to inhibit study, the we expression found that of the genes protein involved levels inof iron-consumingboth Cir1 and HapX functions were si [36gnificantly]. In the current reduced study, in the we sod2 found mutant. that Of the these protein changes, levels re- of bothduction Cir1 of andCir1 HapXmight weretrigger significantly a more detrimenta reducedl effect in the onsod2 the mutant.mutant cells Of thesebecause changes, of the reductionde-repression of Cir1 of genes might involved trigger ain more iron detrimentaluptake, causing effect increased on the mutant accumulation cells because of intra- of thecellular de-repression iron. In mammalian of genes involved cells, overexpression in iron uptake, of mitochondrial causing increased Mn-SOD accumulation significantly of intracellularreduced non-heme iron. In iron mammalian levels, which cells, is overexpressionin agreement with of mitochondrial our data [37,38]. Mn-SOD We hypothe- signif- icantlysize that reduced increased non-heme superoxide iron due levels, to lack which of Sod2 is in may agreement also cause with a ourreduction data [37 in,38 the]. sta- We hypothesizebility and activity that increased of Cir1 and superoxide HapX since due the to proteins lack of Sod2 are predicted may also causeto contain a reduction Fe-S clus- in theters stabilityand iron and influences activity their of Cir1 stability and HapX [21,36]. since Indeed, the proteins previous are studies predicted have to reported contain Fe-Sthat clusters and iron influences their stability [21,36]. Indeed, previous studies have reported

J. Fungi 2021, 7, 523 11 of 13

that superoxide causes the release of iron from the Fe-S cluster and in turn inactivates the Fe-S cluster-containing protein [39,40]. We found that oxidative stress induces vacuole fragmentation in C. neoformans, and deletion of SOD2 exaggerated fragmentation of the organelle. A similar phenomenon was observed in S. cerevisiae, although vacuole fragmentation was mainly observed under hyperosmotic conditions in relation to the transient increase in intracellular levels of PI(3,5)P2, which was synthesized by Fab1 and presented at the vacuole membrane [2,41]. Loss of water under hyperosmotic conditions was hypothesized to reduce the volume of vacuoles and trigger fragmentation of the organelle, and consequently, increase levels of PI(3,5)P2 [2]. In addition to hyperosmotic conditions, oxidative stress also induced vacuole fragmentation in S. cerevisiae. However, unlike C. neoformans, the mutant lacking cytosolic SOD1, but not mitochondrial SOD2, showed increased vacuole fragmentation in S. cerevisiae [6]. Moreover, vacuole fragmentation in the S. cerevisiae sod1 mutant was reduced upon induction of iron deficiency and increased by the addition of excess metal. The vacuole is the main iron storage organelle in S. cerevisiae, and at least two vacuolar iron transport systems have been identified in the fungus. One is mediated by the homologs of the high-affinity plasma membrane iron transport system Fth1-Fet5, whereas the other is mediated by the iron/Mn2+ transporter, Ccc1 [42,43]. Iron storage may be disrupted by the deletion of SOD1 due to defects in correct vacuole morphology in S. cerevisiae [6]. We also speculate that, based on the previous studies and our current findings, vacuole fragmentation increases the surface-to-volume ratio and provides more docking sites for vacuolar iron transporters to increase iron uptake into vacuoles to protect cells from oxidative stress. A positive correlation between the surface-to-volume ratio and increased activity of vacuole transporters was also hypothesized by Michaillat et al. [5]. In general, the regulation of vacuole morphology for C. neoformans may be similar to that of S. cerevisiae. However, Sod2, rather than Sod1, plays a more critical role in vacuole morphology in C. neoformans. Overall, we conclude that the function and morphology of vacuoles are tightly connected to metal homeostasis and oxidative stress responses in C. neoformans. TORC1 is a protein complex that consists of Tor1 or Tor2, Tco89, Kog1, and Lst8, which are mainly localized at the membrane of the vacuoles in S. cerevisiae, and the involvement of TORC1 in vacuole morphology has been documented [44]. The TORC1 pathway has been identified in C. neoformans, and a study confirmed its main localization at the vacuole membrane [45]. Moreover, the influence of the TORC1 pathway on thermotolerance and the DNA damage response in C. neoformans has been suggested [45]. Here, we showed that rapamycin treatment reduced vacuole fragmentation in C. neoformans, suggesting a role for TORC1 in the vacuole morphology of the fungus. Moreover, our study demonstrated that TORC1 and the downstream target protein Fab1 may coordinately regulate vacuole morphology. Interestingly, TORC1 and Fab1 regulation is upstream of oxidative stress- mediated vacuole fragmentation in C. neoformans because the wild-type and the sod2 mutant cells treated with rapamycin under the high concentrations of metals displayed a single large vacuole in our study. In summary, our data demonstrate that the vacuole morphology is significantly influenced by oxidative stress in C. neoformans and that SODs play an important role in mediating the response in the context of metal homeostasis. These aspects of the response to stress are important because an oxidative attack on C. neoformans cells is a key aspect of the innate immune defense mechanisms of host phagocytic cells. This idea is rein- forced by our demonstration that a cytosolic localization of Sod2 occurred in phagocytosed C. neoformans cells, a phenotype that may be associated with metal limitation within a macrophage.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7070523/s1, Table S1: Primers used in this study, Figure S1: Superoxide dismutase activity assay. J. Fungi 2021, 7, 523 12 of 13

Author Contributions: Conceptualization, D.K., E.D., J.W.K. and W.H.J.; methodology, D.K., M.S., E.D., Y.C., J.W.K. and W.H.J.; formal analysis, D.K., M.S., Y.C. and W.H.J.; writing—original draft preparation, D.K., J.W.K. and W.H.J.; writing—review and editing, D.K., J.W.K. and W.H.J.; supervi- sion, J.W.K. and W.H.J.; funding acquisition, J.W.K. and W.H.J. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning 2019R1F1A1061930 (W.H.J.), 2019R1A4A1024764 (W.H.J.), and the National Institute of Allergy and Infectious Diseases RO1 AI053721 (J.W.K.). J.W.K. is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology, and a Canadian Institute for Advanced Research (CIFAR) Fellow in the Fungal Kingdom: Threats and Opportunities Program. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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