Identification of RAVE/Rabconnecntin-3 complex components in C. neoformans

The S. cerevisiae RAVE complex consists of Rav1, Rav2, and Skp1, with Rav1 being the central component [20,24]. Rav1 generally contains a single evolutionarily conserved Rav1 domain and multiple WD40 repeats. We identified a Rav1 ortholog in the C. neoformans genome database (CnRav1). Other fungi, including C. albicans (Ca), Schizosaccharomyces pombe (Sp), and Ustilago maydis (Um), also appear to have a single Rav1 ortholog, with size ranges from 1235 to 1518 amino acids (aa) (Fig 1A). The CnRav1 domain consists of 14 WD40 repeats, which span from the N-terminus to the central region, like other fungal Rav1 orthologs, except for ScRav1 (13 WD40 repeats). Human Rav1 orthologs (Raboconnectin-3α; DMXL1 and DMXL2) are structurally distinct from cryptococcal and other fungal Rav1 orthologs. DMXL1 and DMXL2 have a much larger size (3027 aa and 3036 aa) than fungal Rav1 orthologs and have 10 and 6 WD40 repeats at the N- and C-terminus, respectively, and two Rav1 domains in the central region. Several higher eukaryotes, including humans, have two Rabconnectin-3α subunit isoforms, but other eukaryotes, including Drosophila melanogaster, have only a single Rabconnectin-3α isoform [23]. Like structural differences, CnRav1 was phylogenetically closer to other fungal Rav1 orthologs than hsDMLX1 and hsDMXL2.

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Fig 1. The hRAVE complex is a hybrid of the yeast RAVE complex and the human Rabconnectin-3 complex.

The domain structure and phylogenetic tree of Rav1 and Wdr1/Rav2 orthologs in eukaryotes are illustrated in panels A and B, respectively, with abbreviations indicating the corresponding species: U. maydis (Um), C. neoformans (Cn), C. albicans (Ca), S. cerevisiae (Sc), D. melanogaster (Dm), H. sapiens (Hs), A. thaliana (Ath), S. pombe (Sp), H. capsulatum (Hc), A. fumigatus (Af), M. globosa (Mg), N. crassa (Nc), and F. graminearum (Fg). Domain information for Rav1 and Wdr1/Rav2 was retrieved from InterPro (https://www.ebi.ac.uk/interpro/), UniProt (https://www.uniprot.org/), and WDSPdb 2.0 (https://www.wdspdb.com/wdsp/). The domain IDs for each protein are provided as follows: Rav1 domain (IPR022033; RAVE complex protein Rav1 C-terminal), Rav2 family (IPR028241; RAVE subunit 2/Rogdi), and WD40 repeats (Uniprot KW-0853; WD40 repeat and WDSP-predicted WD40 repeat). (C) The expected model of the hRAVE complex in C. neoformans is shown.

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In contrast to Rav1, C. neoformans does not appear to have any yeast Rav2 ortholog. Instead, the fungal pathogen seems to have an ortholog (CNAG_02402) of Rabconnectin-3β (WDR7), which is structurally and phylogenetically distant from yeast Rav2 (Fig 1B). Interestingly, WDR7 orthologs are present in some fungal species, including U. maydis, Mucor circinelloides, and C. neoformans, but not in ascomycetous yeast species such as S. cerevisiae, S. pombe, and C. albicans (S1 Table). As a previous independent study named CNAG_02402 WDR1 [25], we denoted it as WDR1 instead of WDR7.

Another RAVE component Skp1 ortholog (CNAG_00829), was also present in C. neoformans (S1A Fig). However, Skp1 interacts with various protein complexes in addition to Rav1 of the RAVE complex [20,26,27] (S1B Fig). Supporting it, deletion of SKP1 is not feasible in S. cerevisiae, suggesting that Skp1 is an essential yeast protein. Similarly, our multiple knockout attempts of SKP1 in the haploid C. neoformans H99 strain failed to yield correct knockout mutants. Furthermore, we found that repression of SKP1 expression by replacing its native promoter with the copper-repressible CTR4 (copper transporter 4) promoter resulted in marked growth defects in C. neoformans (S1C and S1D Fig), implying that Skp1 could be a putative essential protein in the pathogen. To more definitively establish the essential role of Skp1 in the viability of C. neoformans, we generated heterozygous SKP1/skp1Δ mutants in AI187, a genetically engineered diploid strain [28] (S1E Fig). Despite examining over 50 spores through random spore analysis, we were unable to isolate any haploid spores with an SKP1 knockout, thereby confirming the essential nature of Skp1 in C. neoformans (S1F Fig). Given these findings and existing reports suggesting that human Skp1 is unlikely to interact with DMXL1 and DMXL2 [23], we opted not to further investigate Skp1’s role in functionally characterizing the RAVE complex.

All these data indicate that C. neoformans appears to have the hybrid form of the yeast RAVE complex and human Raboconnectin-3 complex because CnRav1 seems to be more orthologous to yeast Rav1 in terms of its protein size and structural configuration and CnWdr1, but not CnRav2, is present in C. neoformans (Fig 1C). In this sense, we named it the hybrid RAVE (hRAVE) complex because CnRav1 and CnWdr1 play major and minor roles in diverse pathobiological functions in C. neoformans and interact with each other, as described later.

Roles of the RAVE complex in growth, cell cycle control, and morphogenesis

To elucidate the hRAVE complex’s function, we employed a reverse genetics approach for CnRav1 and CnWdr1. To this end, we generated rav1Δ, wdr1Δ, and wdr1Δ rav1Δ mutants in MATα C. neoformans strain (H99) background through homologous recombination (S2 Fig). To verify their mutant phenotypes, we also generated rav1Δ::RAV1 by reintegrating its wild-type allele into its native locus (S2 Fig). First, we compared the growth of rav1Δ and wdr1Δ mutants with that of the wild-type and complemented strains by spot assays at a varying range of temperature: 25°C, 30°C, 37°C, and 39°C. RAV1 deletion led to a marked growth defect even under nutrient-rich conditions [yeast extract-peptone-dextrose (YPD) medium] at 30°C, whereas WDR1 deletion did not affect the growth (Fig 2A). At elevated temperatures (39°C), rav1Δ showed even more severe growth defects and even wdr1Δ exhibited an evident growth defect at 39°C (Fig 2A). Notably, the growth defect of the rav1Δ wdr1Δ double mutant was as defective as the rav1Δ single mutant (Fig 2A), suggesting that Rav1 and Wdr1 may play central and accessary roles, respectively, in the hRAVE complex. Quantitative growth rate measurement further confirmed its growth patterns (Fig 2B).

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Fig 2. The role of Rav1 in growth, cell cycle control, and morphological change.

(A) Qualitative spot assays showing growth and thermosensitivity of WT (H99), rav1Δ (YSB7589), rav1Δ::RAV1 (YSB10754), wdr1Δ (YSB10032), wdr1Δ rav1Δ (YSB10604) strains. Plates were incubated for 2 days and photographed. (B) Quantitative growth rates of the same strains monitored at 30 °C, 37 °C, and 39 °C using a multi-channel bioreactor for 40 h. Each curve is a single representative of two independent experiments. (C, D) Flow cytometry analysis of cell cycle phases in propidium iodide (PI)-stained cells. Representative data from three biological replicates are shown. (D) The percentage of cell populations classified as interphase (G₁ peak and S phase) and mitotic phase (G₂/M peak) from the same experiment (C) was quantified and presented in the graph. One-way ANOVA with Bonferroni’s multiple-comparison test determined statistically significant differences between the wild-type and mutants. Error bars indicate the standard error of the mean (SEM) (NS, non-significant; **, P = 0.001–0.01). (E) Quantification of unseparated cells measured for each C. neoformans strain cultured on YPD, Littman, DME, and RPMI agar media at 37°C for 2 days. After picking a colony from the media, cells were washed twice with PBS, added with 0.5 ml PBS, and briefly sonicated. Capsules were stained with India ink, and the morphology of the wild-type and mutant strains were observed under a microscope (Bar, 10 μm). More than 100 cells were measured, and the graph shows the average of three biological replicates.

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Apparent growth defects observed in rav1Δ strains implied that the hRAVE complex is likely involved in the cell cycle control of C. neoformans. Supporting this, fluorescence-activated cell sorting (FACS) analysis revealed that distinct cell cycle phases observed in the wild-type were absent in rav1Δ (Fig 2C). Unlike the wild-type cells, which had discrete n and 2n cell populations, the rav1Δ cells showed an aberrant ploidy distribution. In addition, the cell ratio of the G₂/ M phase was much lower in the rav1Δ mutant than in the wild-type strain (Fig 2D).

Defective growth and cell cycle control of the rav1Δ mutant led us to suspect that the hRAVE complex could be involved in the morphogenesis of C. neoformans. Therefore, we observed the cellular morphology of rav1Δ in nutrient-rich solid medium (YPD) and capsule-inducing solid media (Littman, DME, and RPMI). We briefly sonicated cultured cells and observed their morphology to discern whether such cell aggregation resulted from mere physical association among cells or cytokinesis defects. The rav1Δ cells were more aggregated than wild-type ones (upper panel in Fig 2E). Quantitative measurement confirmed this finding (lower panel in Fig 2E). In the wild-type, the vast majority of cells (>80%) only exist as single cells, and there were no four or more linked cells. In wdr1Δ, 40–70% of cells exist as single cells, and 5–20% of four or more connected cells exist. Yet, in rav1Δ, fewer than 20% of cells exist as single cells, and roughly 40–80% of four or more connected cells exist (Fig 2E). We also made a similar finding in broth media (S3A Fig). Interestingly, when cultured in Littman, DME, and RPMI media, rav1Δ cells showed pseudohyphal growth (upper panel in Fig 2E). In contrast to rav1Δ, most wdr1Δ cells exhibited similar cellular morphologies to the wild-type in the YPD medium, but some showed somewhat elongated cell morphology in the capsule-inducing media.

The RAM signaling pathway is highly conserved among eukaryotes and plays a role in diverse cellular processes, including cell cycle regulation, cell separation, mating, and cell polarized growth [29]. Unusual morphological changes of rav1Δ, which seem like pseudohyphal growth, allowed us to investigate a link with the RAM pathway related to pseudohyphal development in C. neoformans [30]. We compared rav1Δ with cbk1Δ and kic1Δ in the RAM pathway in several solid growth and capsule-inducing media. Both RAM pathway mutants showed pseudohyphal growth in all media, whereas rav1Δ showed pseudohyphal growth only in capsule-inducing media (Littman, DME, RPMI, FBS) and not in YPD and YNB media (S3B Fig). In addition, the pseudohyphal form of rav1Δ also differed from that of RAM pathway mutants: cbk1Δ and kic1Δ were long rod-shaped, but rav1Δ was more rounded and shorter. This indicates that Rav1 is involved in morphological changes, and its role appeared independent of the RAM pathway.

These results suggested that the hRAVE complex, in which Rav1 and Wdr1 are the central and accessory components, respectively, is required for the growth, cell cycle control, and morphogenesis of C. neoformans.

Roles of the hRAVE complex in the sexual development of C. neoformans

The function of the hRAVE complex in the morphogenesis of C. neoformans prompted us to address its roles in yeast-to-filament morphological transition during the sexual differentiation of the pathogen. To this end, we additionally generated rav1Δ and wdr1Δ mutants in the MATa strain (YL99) background (S4A Fig). In both unilateral (wild-type x mutant) and bilateral (mutant x mutant) mating with rav1Δ, filamentation was abolished entirely (Fig 3A). The fact that rav1Δ mutants did not show any filamentous growth even after 25 days of incubation on the V8 medium suggested that this result is not simply due to the growth defect of the rav1Δ strain. In support of this, the expression of RAV1 increased about ten folds at 24 h postmating, implying that RAV1 could be involved in the mating process (Fig 3B). Moreover, the wdr1Δ, which had no growth defect at 25°C, showed reduced filamentation in both unilateral and bilateral mating (S4B Fig), albeit weaker than the phenotype of rav1Δ, further strengthening the claim that the hRAVE complex could be involved in the mating process.

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Fig 3. The role of Rav1 in sexual differentiation.

(A) Qualitative photographs of cocultured MATα and MATa strains on V8 medium plates (pH 5.0) for 25 days at room temperature in the dark. Strains used for the mating assay are: α (H99) × a (YL99), α rav1Δ × a (YL99a), α (H99) × a rav1Δ, and α rav1Δ × a rav1Δ. (B) qRT-PCR analysis of the RAV1 gene during the mating process. (C) qRT-PCR analysis of MFα1 pheromone gene during the mating process. The expression level of each gene was normalized by that of ACT1. (D) Data from cell fusion assay. Control strains containing different selectable markers [MATα YSB119 (NAT) and MATa YSB121 (NEO)] were used. The plate picture is a representative of three biological replicates. Percentages normalized to the positive control are mean values from each experiment indicated by a dot. The error bars indicate the standard errors of the means (SEM). Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple-comparison test: ***, P < 0.0001.

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Next, we addressed which mating step the hRAVE complex is involved in. First, we measured the expression level of the MFα1 pheromone gene during bilateral mating (Fig 3C). The MFα1 expression increased about 15 folds at 24 h and 48 h postmating of MATα and MATa wild-type cells. In bilateral mating with rav1Δ mutants, however, MFα1 expression did not significantly increase after 24 h and 48 h postmating (Fig 3C). Such defective pheromone induction during mating with rav1Δ mutants could affect cell-to-cell fusion. As expected, unilateral mating between the control and rav1Δ strain led to a significantly reduced cell fusion at 24 h and 48 h postmating. Bilateral mating with rav1Δ mutants led to a complete loss of cell fusion (Fig 3D). All these data suggest that the hRAVE complex is required to activate the early mating stage in C. neoformans.

Roles of the hRAVE complex in cryptococcal virulence factor production

Next, we investigated whether the hRAVE complex is involved in producing two major virulence factors in C. neoformans: antiphagocytic polysaccharide capsule and antioxidant melanin pigment [31,32]. As shown in Fig 2E, the rav1Δ mutant appeared to produce wild-type capsule levels in most capsule-inducing media but slightly decreased in the Littman medium. However, quantitative capsule size measurement was not feasible due to the elongated, pseudohyphae-like morphology of the mutant. Therefore, we addressed whether Rav1 regulates the expression of capsule-related genes, such as CAP10, CAP59, CAP60, CAP64, GAT201, YAP1, ADA2, and BZP4, in Littman medium (S5A Fig). Although Rav1 did not significantly regulate the expression of most capsule-related genes, the expression of capsule-regulating transcription factor GAT201 significantly increased under Littman-based capsule-inducing conditions in the rav1Δ mutant. As we recently reported that GAT201 overexpression markedly enhances capsule production [33], GAT201 expression appeared to be induced as a compensatory mechanism in the rav1Δ mutant.

In contrast to the minor role in capsule production, the hRAVE complex played a critical role in melanin production. The rav1Δ mutant showed even more severe defects in melanin production than the adenylyl cyclase mutant (cac1Δ) in all melanin-inducing media (Fig 4A). Quantitative measurement of laccase activity further confirmed this finding (Fig 4B). Moreover, melanin production of the wdr1Δ was also reduced compared to the wild type, indicating that the hRAVE complex is involved in melanin production (S5B Fig).

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Fig 4. The role of Rav1 in melanin production.

(A) Melanin production assay. The wild-type (WT; H99), rav1Δ (YSB7465 and YSB7589), rav1Δ::RAV1 (YSB10754 and YSB10755), hog1Δ (YSB64), and cac1Δ (YSB42) mutants were spotted onto Niger seed, epinephrine, or L-DOPA agar medium containing 0.1% glucose, incubated at 37°C for 3 days, and then photographed. Positive and negative controls were hog1Δ and cac1Δ strains, respectively. Representative images from three independent experiments are shown here. (B) Laccase assays. WT (H99), rav1Δ (YSB7589), rav1Δ::RAV1 (YSB10754), and lac1Δ (CHM3) mutants were cultured in liquid YPD medium overnight, washed thrice with PBS, and incubated for 4 days in YNB plus 0.05% glucose with 10 mM epinephrine. (C) qRT-PCR analysis of melanin production-related genes, LAC1, USV101, MBS1, HOB1, and BZP4, using total RNA of WT and rav1Δ mutant strains under nutrient-rich (R; YPD) or nutrient-starved (S; YNB without glucose) conditions. For induction of melanin production-related genes by nutrient starvation, cells were further incubated at 30°C in YNB media for 2 h. One-way ANOVA with Bonferroni’s multiple-comparison test was used to determine the statistical significance of differences: *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, non-significant. Error bars indicate SEM.

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The rav1Δ mutant was as defective in melanin production as the mutant with deleted LAC1, which encodes a central melanin-biosynthetic laccase enzyme in C. neoformans [34]. We next addressed whether Rav1 is involved in the induction of LAC1 and melanin-regulating transcription factors, USV101, MBS1, HOB1, and BZP4, under nutrient-starvation conditions (Fig 4C). In contrast to highly defective melanin production in rav1Δ, the expression of LAC1, MBS1, HOB1, and BZP4 was overall increased under glucose-starved conditions in rav1Δ compared to wild-type (Fig 4C). All these data suggested that the hRAVE complex does not directly regulate melanin biosynthesis pathways and its inhibition may induce the melanin-regulating genes as a compensatory mechanism.

Roles of the hRAVE complex in organelle acidification and vesicle trafficking

It is well known that the yeast RAVE complex is involved in organelle acidification and trafficking of the trans-Golgi network (TGN) and secretory/endocytic vesicles [13,22,24,35]. To address whether the hRAVE complex plays similar roles in C. neoformans, first, we monitored the cellular location of Rav1 by generating rav1Δ::RAV1-mCherry complemented strains, in which mCherry was in-frame fused to the C-terminus of Rav1. The rav1Δ::RAV1-mCherry strain was phenotypically identical to wild-type and rav1Δ::RAV1 strains, indicating that Rav1-mCherry is functional (S6A and S6B Fig). When we stained the Rav1-mCherry strain with the LysoSensor dyes, which are acidotropic probes that accumulate in acidic organelles as the result of protonation, Rav1 appeared to localize to the cytosol and to be particularly enriched in the vacuole periphery as punctate forms (Fig 5A). Notably, such punctate signal intensity of Rav1-mCherry around the vacuole membrane increased at high glucose concentration, which agrees with increased acidification via high glucose levels like the case of S. cerevisiae [36]. All these data indicate that C. neoformans undergoes organelle acidification upon glucose addition, probably through the hRAVE complex.

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Fig 5. The role of the hRAVE complex in vesicle trafficking.

(A) Acidification monitoring in Rav1-mCherry strain under different glucose concentrations. The strain was cultured in YPD overnight and then inoculated in liquid YNB medium without glucose (Starvation), with glucose concentrations of 5 mM, 25 mM, or 250 mM for one hour. Cells cultured without glucose (Starvation) were replaced with fresh YNB containing 250 mM glucose (Readdition) and cultured again for one hour. Acidification was measured using LysoSensor dyes as an acidotropic probe. Fluorescence microscopy was used to observe cells under each condition. One representative image from three biological replicates is shown here. Bars indicate 10 μm. (B) Localization of Ste6 in response to deletion of RAV1 during mating. FM4-64 was used as a vacuole membrane-staining dye. All images are representative images of more than 50 cells observed and photographed. (C) Transmission electron microscopy (TEM) analysis of WT and rav1Δ mutant strain. The black arrow indicates the vacuole. Both images are shown at 150 k magnification.

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The acidification of organelles is closely linked to protein sorting and organelle function [36]. Ligand dissociation from their respective receptors takes place within a specific pH range, which serves a crucial role in determining their final targeting [1]. Furthermore, the luminal pH of endosomes can impact the association of trafficking factors [37]. For these reasons, we monitored the impact of RAV1 deletion in the cellular localization of the pheromone transporter Ste6. In S. cerevisiae, the membrane localization of Ste6 is controlled by endosome trafficking [38]. We also previously reported that cryptococcal Ste6 is localized to ER and endosomes under vegetative growth conditions but highly enriched at the plasma membrane upon switching to the mating condition [39]. Therefore, we deleted RAV1 in the previously constructed STE6-GFP strain (S7 Fig). We hypothesized that RAV1 deletion could cause defects in normal vesicle trafficking of Ste6 to the plasma membrane. Therefore, we cultured STE6-GFP and STE6-GFP rav1Δ strains in YPD medium for vegetative growth and V8 medium for mating and stained them with the lipophilic styryl dye FM4-64 that binds to the plasma membrane and is then endocytosed and trafficked to the vacuolar membrane [40] (Fig 5B). As expected, after 24 h postmating, Ste6 localized to the cell membrane in STE6-GFP strains. However, in STE6-GFP rav1Δ strains, Ste6 appeared to be accumulated in the vacuole, not the cell membrane, even under mating conditions (Fig 5B). This phenomenon can explain why RAV1 deletion abolished the mating of C. neoformans (Fig 3) because Ste6 is required for the extracellular release of pheromone that initiates the mating process.

To support these results, we performed transmission electron microscopy (TEM) for wild-type and rav1Δ cells. In comparison to the wild-type strain, the rav1Δ strain frequently demonstrated an accumulation of vesicles that increased the size and the number of vacuoles (Fig 5C). These observations support previous findings that the hRAVE complex regulates the vesicle trafficking of Ste6. Furthermore, defective melanin production in rav1Δ shown in Fig 4 also likely resulted from the perturbed vesicle trafficking of melanin-containing vesicles to the cell surface. All these data indicate that the hRAVE complex is essential for organelle acidification and vesicle trafficking in C. neoformans.

Intracellular pH-dependent and -independent roles of the hRAVE complex

In yeast, deletion of any V₀ or V₁ subunits of V-ATPase leads to a very similar phenotype, called Vma^- phenotype, with pH and calcium-sensitive growth, metal ion sensitivity, and inability to grow on non-fermentable carbon sources [3]. pH-dependent growth is one of the representative Vma^- phenotypes, in which V-ATPase mutants are viable when grown in acidic media but cannot grow in alkaline conditions. It is also known that yeast RAVE complex mutants exhibit a partial Vma^- phenotype, which is temperature-dependent and milder than in a V-ATPase mutant; yeast rav1Δ mutant shows growth defect at 37°C, but very slightly at 30°C, on pH 7.5-adjusted YPD medium [20,24]. However, we found that the C. neoformans rav1Δ mutant exhibited growth defects even at 30°C on pH 7.5-adjusted YPD medium but significantly restored growth on pH 5.5-adjusted YPD medium (Fig 6A and 6B). This result indicated that deletion of the hRAVE complex results in a more evident Vma^- phenotype (e.g., pH-dependent growth) than the yeast RAVE complex.

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Fig 6. Phenotypic analysis of pH-dependent or -independent function of the hRAVE complex.

(A) Qualitative spot assays under various temperatures (25°C, 37°C, and 39°C) and pH levels (pH 5.5, pH 6.8, and pH 7.5). (B) Quantitative growth rates of the WT and rav1Δ strains on different pH levels. Growth was monitored at 30°C by measuring OD₆₀₀ with a multi-channel bioreactor for 50 h. Each curve represents a single trial of two independent experiments. (C) Qualitative spot assays under indicated stress conditions at different pH levels. Plates were incubated at 30°C for 3 days. Abbreviations: TBH (tert-butyl hydroperoxide), 0.7 mM; MD (menadione), 0.02 mM; MMS (methyl methanesulphonate), 0.03%; HU (hydroxyurea), 100 mM; CR (Congo red), 0.8%; CFW (calcofluor white), 5 mg/ml; CDS (cadmium sulfate), 25 μM; AMB (amphotericin B), 1.6 μg/ml; H₂O₂ (hydrogen peroxide), 3 mM; DIA (diamide), 2.5 mM; TM (tunicamycin), 0.3 μg/ml; DTT (dithiothreitol), 16 mM; SDS (sodium dodecyl sulfate), 0.03%; SBR (YPD + 2 M sorbitol); FCZ (fluconazole), 10 μg/ml; FDX (fludioxonil), 1 μg/ml; 5FC (5-flucytosine), 300 μg/ml. Blue and red fonts indicate pH-dependent and -independent phenotypes, respectively.

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Next, we further investigated the pH-dependent growth phenotypes of the hRAVE complex mutants under various stress conditions. For this purpose, we compared the growth of wild-type, rav1Δ, and rav1Δ::RAV1 strains in YPD growth media having different stress-inducing agents under varying pH ranges (5.5, 6.8, and 7.5) (Fig 6C). Notably, we found that acidic pH adjustment of the growth medium differentially restored the resistance of rav1Δ to various stress conditions. First, acidic pH adjustment markedly restored the growth of rav1Δ in a YPD medium containing certain oxidative stress agents (tert-butyl hydroperoxide and menadione), genotoxic agents (methyl methanesulfonate and hydroxyurea), cell wall destabilizing agents (congo-red and calcofluor white), heavy metal stress agent (CdSO₄), or a polyene class of antifungal drug (amphotericin B) (Fig 6C). In contrast, acidic pH adjustment did not rescue or only weakly restored the growth defects in the YPD medium containing hydrogen peroxide (H₂O₂), thiol-specific oxidant (diamide), ER stress agents (tunicamycin and DTT), membrane-destabilizing agent (SDS), osmotic stress agent (sorbitol), or non-polyene classes of antifungal agents (fluconazole, fludioxonil, and flucytosine) (Fig 6C). All these data indicate that the hRAVE complex is likely involved in both pH-dependent and pH-independent phenotypes in C. neoformans.

V-ATPase-dependent and -independent roles of the hRAVE complex

We aimed to discern whether the hRAVE complex operates through two distinct pathways: V-ATPase/pH-dependent and -independent mechanisms. Specifically, we focused on its influence on growth, morphogenesis, sexual differentiation, melanin production, and various stress responses and adaptations. To establish a direct association with V-ATPase, we treated wild-type with a specific V-ATPase inhibitor, bafilomycin A1. The concentration of bafilomycin A1 utilized in this study was 500 nM, which has been previously reported to specifically inhibit V-ATPase at this dose [41,42]. By treating the wild-type strain with this inhibitor, we aimed to determine if it could lead to rav1Δ-like phenotypic changes in wild-type C. neoformans. First, bafilomycin A1 can cause defects in the growth of C. neoformans at the temperature range of 25°C to 39°C (Fig 7A). Second, bafilomycin A1 treatment caused elongated, pseudohyphae-like cellular morphology in wild-type C. neoformans in a dose-dependent manner (Fig 7B). Third, bafilomycin A1 treatment inhibited mating of wild-type MATα and MATa strains at 10 days postmating on V8 medium, although its inhibition effects disappeared at 25 days postmating (Fig 7C). Fourth, bafilomycin A1 treatment completely abolished melanin production in C. neoformans, which is almost equivalent to the effect of LAC1 deletion (Fig 7D). All these results indicate that the hRAVE complex plays pleiotropic roles in growth, morphogenesis, sexual development, and melanin production in C. neoformans via V-ATPase-dependent pH regulating functions.

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Fig 7. Phenotypic analysis of V-ATPase-dependent or -independent function of the hRAVE complex.

(A-E) Wild-type phenotypic traits in response to bafilomycin A1 treatment (500 nM). (A) Temperature-dependent stress response of wild-type. The plates were incubated at 30°C for 2 days. (B) Morphological change of wild-type strain. The capsule was stained with India ink and observed by microscopy (Bar, 10 μm). More than 50 cells were observed and photographed. (C) Mating assay for bafilomycin A1 treatment in early and late phases. α H99 × a YL99 strains were cocultured on V8 medium with or without bafilomycin A1 for 10 to 25 days at room temperature in the dark. One representative from three biological replicates is shown here. (D) Laccase assay for bafilomycin A1 treatment. WT (H99) and lac1Δ (CHM3) mutants were incubated for 4 days in the YNB medium (see Fig 4B) supplemented with bafilomycin A1. (E) Phenotypic traits of WT treated with bafilomycin A1 under stress conditions described in Fig 6C. The plates were incubated at 30°C for 3 days. Blue and red fonts indicate V-ATPase-dependent and -independent phenotypes, respectively.

https://doi.org/10.1371/journal.ppat.1011721.g007

Finally, we examined the effect of bafilomycin A1 on the resistance of C. neoformans against various stress-inducing agents. In agreement with the data presented in Fig 6C, the effect of bafilomycin A1 treatment varied with different kinds of stress-inducing agents. Under most stresses, bafilomycin A1-treated wild-type cells exhibited reduced growth rates compared to controls, indicating that the hRAVE complex is involved in stress response and adaptation in a predominantly V-ATPase-dependent manner. On the other hand, upon exposure to several stressors, including fluconazole, H₂O₂, menadione, diamide, and hydroxyurea, the growth rate of the wild-type strain treated with bafilomycin A1 exhibited no significant difference from the control group (Fig 7E). Notably, the increased susceptibility of rav1Δ to fluconazole and H₂O₂ also appeared to be pH-independent phenotypes (Fig 6C), suggesting that the hRAVE complex may play a novel role in stress response and adaptation beyond its well-established function as a regulator of V-ATPase activity. To further elucidate the V-ATPase-dependency of the hRAVE complex, we conducted the same experiments on the rav1Δ mutant (S8A Fig). In most V-ATPase-dependent phenotypes, the growth rate of the rav1Δ mutant treated with bafilomycin A1 showed no significant difference from the untreated control, indicating that rav1Δ plays a crucial role as a regulator of V-ATPase assembly. However, under certain stresses, such as amphotericin B, tert-butyl hydroperoxide, calcofluor white, and hydroxyurea, we observed further growth inhibition in the rav1Δ mutant upon bafilomycin A1 treatment. This suggests the possible involvement of other regulators in V-ATPase assembly under these specific stresses in C. neoformans.

Furthermore, our findings corroborate that these V-ATPase-independent stress responses are modulated either through a Wdr1-dependent pathway, as observed with menadione or diamide treatment, or via Wdr1-independent pathway, evident with fluconazole, hydrogen peroxide, or hydroxyurea treatment (S8B Fig). Therefore, in the context of V-ATPase-independent stress responses, Rav1 and Wdr1 may collaborate or Rav1 can function independently. All these data indicate that the hRAVE complex plays V-ATPase/pH regulation-dependent and -independent roles in C. neoformans.

Proteomics analysis for identifying hRAVE complex-interacting proteins

The finding that the hRAVE complex plays V-ATPase/pH-dependent and -independent roles in C. neoformans prompted us to search for other hRAVE-interacting partners. To this end, we performed in vivo pull-down experiment using the rav1Δ::RAV1-mCherry strain and RFP-trap agarose beads to precipitate Rav1-mCherry protein and its interacting partners. As expected, Wdr1 (CNAG_02402) was identified as one of the Rav1-interacting proteins (S2 Table), further supporting that Rav1 and Wdr1 likely constitute the hRAVE complex, as we predicted. Furthermore, one of the V-ATPase subunits (Vma1; CNAG_02326) was also identified (S2 Table), suggesting that the hRAVE complex interacts with V-ATPase. Interestingly, Rav1 appeared to interact with α (CNAG_05750) and β (CNAG_05918) subunits of F-type ATPase, suggesting that the hRAVE complex could control other ATPases.

V-ATPase has been reported to interact with several glycolytic enzymes, including aldolase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase [43,44]. We also found that several glycolysis-related proteins were potential Rav1 interactors in C. neoformans: fructose-bisphosphate aldolase (CNAG_06770), glyceraldehyde-3-phosphate dehydrogenase (CNAG_06699), phosphoglycerate kinase (CNAG_03358), phosphopyruvate hydratase (CNAG_03072), pyruvate kinase (CNAG_01820), pyruvate decarboxylase (CNAG_04659). Furthermore, functional categories of Rav1-interacting proteins include amino acid biosynthesis: 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (Met6; CNAG_01890) and aspartate carbamoyltransferase (CNAG_07373). Stransky et al. demonstrated that amino acids regulate the activity and assembly of V-ATPase [45], indicating that the methionine and aspartate synthesis pathway may function with the RAVE complex. All these data implied that the hRAVE complex could interact with glycolytic and amino acid biosynthetic enzymes to regulate V-ATPase assembly.

Besides the Rav1-interacting proteins implicated in the V-ATPase-dependent roles of the hRAVE complex, the following Rav1-interactors appeared to be involved in the V-ATPase-independent functions of the hRAVE complex. Transaldolase (Tal1; CNAG_01984) was identified to be one of the Rav1-interacting proteins (S2 Table). Tal1 is involved in the pentose phosphate pathway in yeast [46] and controls oxidative stress response and mitochondrial function in humans [47]. Consistent with these data, the deletion of RAV1 exhibited severe V-ATPase-independent defects on oxidative stress (Fig 6C). These proteomics data support the result of novel functions of the V-ATPase-independent hRAVE complex and indicate that specific stress responses may be modulated through Rav1-Tal1 interactions.

Roles of the hRAVE complex for the pathogenicity of C. neoformans

To investigate the role of the hRAVE complex in the pathogenicity of C. neoformans, we used wild-type, rav1Δ, and complemented strains in insect and murine models of systemic cryptococcosis. Our results showed that the virulence of the rav1Δ mutant was significantly attenuated compared to the wild-type and complemented strains in both insect (Fig 8A) and murine host models (Fig 8B). Further analysis of fungal burden in infected organs recovered from sacrificed mice revealed that all the rav1Δ-infected organs had reduced fungal burden compared to the wild-type and complemented strain-infected organs (Fig 8C). Additionally, the histopathological analysis showed that rav1Δ cells penetrated the lung tissue less efficiently than the wild-type and complemented cells, with the former observed in a clustered form inside the lung’s bronchioles (Fig 8D). Our finding suggest that Rav1 is essential for C. neoformans to survive, proliferate and invade lung tissues effectively during infection, and may control virulence by altering the ability to transverse the blood-brain barrier (BBB). Indeed, the rav1Δ mutant completely lost the ability to cross the BBB (Fig 8E). In addition, wdr1Δ and wdr1Δ rav1Δ mutants also exhibited severe defects in BBB crossing (S9 Fig), indicating that the hRAVE complex is critical for BBB transmigration. These results suggest that the hRAVE complex is required for the pathogenicity of C. neoformans.

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Fig 8. The role of Rav1 in the pathogenicity of C. neoformans.

(A) Galleria mellonella insect killing assay (n>15) and (B) survival assay in BALB/c murine infection model (n = 7). Statistical differences between the WT (H99) and rav1Δ strain or between WT and +RAV1 complemented strains were calculated using the log-rank (Mantel–Cox) test to measure. (C) Fungal burden assay (n = 5). Mice were sacrificed on day 13 post-infection. Statistical differences were calculated using the one-way ANOVA test. (D) Grocott’s methenamine silver (GMS) staining of lung tissues 14 days after infection. The scale bar indicates 20 μm. (E) BBB crossing assay. The number of yeast cells that either transcytosed through or did not transcytose through the hCMEC/D3-coated Transwell was quantified using colony-forming units (CFU) for each category. The BBB crossing efficiency was calculated as outlined in the Materials and Methods section. The left Y-axis represents the relative BBB crossing ratio, which indicates the normalized BBB crossing efficiency for each tested strain relative to the wild-type (WT) strain. The right Y-axis displays the trans-endothelial electrical resistance (TEER). C. neoformans sit4Δ mutant were used as negative controls. Statistical significance was determined using one-way ANOVA with Tukey’s multiple-comparison test: *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, not significant. Error bars indicate SEM.

https://doi.org/10.1371/journal.ppat.1011721.g008

Ethics statement

Animal care and research were approved after deliberation by the Institutional Animal Care and Use Committee of the Experimental Animal Center at Jeonbuk National University. (Approval number JBNU 2022–092) All experiments followed the experimental ethics guidelines.

Strains, media, and growth conditions

C. neoformans strains used in this study are listed in S4 Table in the supplemental material. Strains were maintained and cultured in 1% yeast extract-2% peptone-2% dextrose (YPD) broth medium or on plates containing 2% agar in YPD broth. Niger seed, _L-3,4-dihydroxyphenylalanine (_L-DOPA), or epinephrine agar medium for melanin production assay and liquid epinephrine medium for laccase assay; agar-based Dulbecco’s modified Eagle (DME), Roswell Park Memorial Institute (RPMI) 1640, and Littman’s media for capsule production assay; and V8 medium containing 5% V8 juice for mating were made as previously described [72].

Construction of rav1Δ and wdr1Δ mutants and complemented strains

To generate rav1Δ and wdr1Δ mutants, we employed homologous recombination in the C. neoformans serotype A MATα wild type strain (H99) and MATa strain (YL99a) backgrounds, respectively. We used gene disruption cassettes containing the nourseothricin resistance marker (NAT) and neomycin resistance marker (NEO). Mutants were constructed according to previously described methods [73]. Targeted gene deletion was confirmed by Southern blot analysis (see S2 Fig in the supplemental material). To validate the observed phenotypes of the rav1Δ mutant, we constructed non-tagging complemented strain via the Gibson assembly method. The full-length gene fragment was amplified via Phusion PCR using the H99 genomic DNA as the template. The amplified fragments of the MATα RAV1 gene were cloned with pNEO plasmid. To observe the localization of Rav1, we also constructed fluorescence (mCherry)-tagged complemented strain by cloning the amplified RAV1 insert into pNEO_mCherry plasmid. After confirming the integration of the target gene into each plasmid via enzyme digestion and sequencing analysis, targeted reintegration into the native locus was performed through biolistic transformation. Plasmids containing the RAV1 gene were linearized by restriction enzyme digestion (NdeI) and introduced into the rav1Δ mutant strain through biolistic transformation. The correct targeted integration of the complemented strains was confirmed via diagnostic PCR.

Construction of heterozygous SKP1/skp1Δ mutants and random spore analysis

For the generation of heterozygous SKP1/skp1Δ mutants, we utilized homologous recombination techniques in the genetically engineered diploid C. neoformans strain AI187 (ade2/ADE2 ura5/URA5 MATa/MATα). Gene disruption cassettes containing the nourseothricin resistance marker (NAT) were used. The knockout construction and validation protocol follow those established for haploid knockout strains. Targeted gene deletion was verified through Southern blot analysis (see S1E Fig in the supplemental material). For sporulation induction, the constructed SKP1/skp1Δ mutants were cultured overnight in liquid YPD medium at 30°C. We spotted 3 μl of 10⁷ cells/ml onto V8 mating medium (pH 5) and incubated the plates in the dark at room temperature (25°C) for over 30 days. For spore isolation, we followed the protocol previously reported by Frerichs et al. [74] with a slight modification. Following incubation on the mating medium, spores were harvested by scraping and resuspended in 75% Percoll buffer (Sigma-Aldrich, P1644). Spore suspensions were vigorously vortexed for 10 seconds until fully resuspended. Subsequently, samples were centrifuged at 3,000 rpm (1,977 x g) at 4°C for 30 min. The bottom of the tube was sterilized with 70% ethanol, and a needle was employed to puncture it. Five drops were collected, followed by the addition of 1.2 ml of PBS. Tubes were centrifuged again for 5 min at 3,000 rpm, and the supernatant was discarded, retaining 100 μl of buffer. An additional 1.4 ml of PBS was added for washing, and this step was repeated twice. For spore plating and verification, spores were then diluted at a 1/100 ratio, and 50 μl of this diluted spore suspension was plated onto YPD medium supplemented with 5-fluoroorotic acid (5-FOA; 100 μg/ml), adenine (20 μg/ml), and uracil (40 μg/ml). Plates were incubated at 30°C for 3 days, after which emerging colonies were streaked onto YPD plates containing chloramphenicol. After 2 days, isolated spores were spotted on various media, including YPD, YPD with nourseothricin (100 μg/ml), YNB, YNB with uracil, YNB with adenine, and YNB with both uracil and adenine. To confirm the haploid status and mating types, diagnostic PCR targeting the mating locus was performed on isolated spores. For those grown in nourseothricin-supplemented YPD, diagnostic PCR was conducted to confirm the successful internal gene deletion.

Measurement of the number of connected cells

Each strain was incubated overnight in liquid YPD medium at 30°C. The cells were washed twice with PBS, resuspended in 0.5 ml of PBS, and spotted (3 μl) onto YPD, Littman, or RPMI agar media. The plates were incubated at 37°C for 2 days. After incubation, a single colony was selected from each plate and suspended in PBS. The cells were then washed twice in PBS and sonicated using a VCX 130 (6mm tip; SONICS, Japan) with a CV18 converter (Vibra-Cell). Sonication was performed for 10 seconds at an amplitude of 20%. Capsule visualization was performed by staining with India ink, and morphology was observed by microscopy (Bar, 10 μm). Over 50 cells were observed and photographed for each wild-type and mutant strains.

Growth and stress sensitivity assays

Each strain was incubated overnight in liquid YPD medium at 30°C with shaking at 220 RPM. The cultures were then serially diluted (1 to 10⁴ dilutions) in distilled water (dH₂O), and spotted (3 μl) onto a solid YPD medium. To assess the growth of C. neoformans strains at different temperatures, serially diluted cells were spotted on YPD agar medium and incubated at 25°C, 37°C, and 39 °C for 1–5 days, with daily photographs taken. To quantitatively examine the growth rate of the rav1Δ mutant, the WT strain (H99) and rav1Δ mutant were incubated at 30°C overnight and subcultured in a fresh liquid YPD medium [optical density at 600 nm (OD₆₀₀) = 0.2]. The cells were then incubated in a multi-channel bioreactor (Biosan Laboratories, Inc., Warren, MI, USA) at 30°C, 37°C, and 39°C, and OD₆₀₀ was automatically measured for 40–50 h. To investigate the response of rav1Δ mutant to various stresses, we spotted onto the YPD medium containing the indicated concentration of the following chemical agents: antifungal drug (amphotericin B, fludioxonil, fluconazole, and flucytosine); oxidative stress (hydrogen peroxide [H₂O₂], tert-butyl hydroperoxide [an organic peroxide], menadione [a superoxide anion generator], diamide [a thiol-specific oxidant]); osmotic stress (sorbitol) under glucose-rich (YPD) condition; genotoxic stress (methyl methanesulfonate and hydroxyurea); cell wall destabilizing stress (calcofluor white and Congo red); membrane destabilizing stress (sodium dodecyl sulfate [SDS]); ER stress (tunicamycin and dithiothreitol [DTT]); heavy metal stress (cadmium sulfate [CdSO₄]). Cells were incubated at 30°C for 1–5 days with daily photographs taken. To analyze stress responses according to the pH of the rav1Δ mutant, the same stress agents were added to YPD media adjusted to pH 5.5, 6.8, and 7.5 using 50 mM MES (for pH 5.5) or 150 mM HEPES (for pH 6.8 or 7.5).

In vitro virulence factor production assays

For melanin production assay, each strain was incubated for 16 h at 30°C in YPD medium, washed three times with PBS, and spotted onto Niger seed, _L-DOPA, or epinephrine agar medium containing 0.1% glucose. The cells were incubated at 37°C for 3 days with daily monitoring and photography of melanin production. Laccase activity was measured quantitatively, as previously described [75]. Briefly, each strain was incubated in 50 ml of YPD medium at 30°C for 16 h. The incubated cells were washed three times with PBS, adjusted to an OD₆₀₀ of 0.2, and inoculated into YNB plus 0.05% glucose supplemented with 10 mM epinephrine. The cultures were incubated at 30°C for 4 days. After incubation, cells were centrifuged, and the OD₄₇₅ of the supernatant was measured with a spectrophotometer. For capsule production assay, each strain was incubated in liquid YPD medium at 30°C for 16 h, spotted onto agar-based DME, Littman, or RPMI medium, and further incubated for 48 h at 37°C. Capsule production was observed by staining with India ink and examining it under a microscope. Microscopy images were captured using a Nikon Eclipse Ni microscope. Unfortunately, quantitative measurement of capsule size was not possible due to the abnormal morphology of the rav1Δ mutant.

Mating and cell fusion assays

Mating and cell fusion efficiency was measured as previously described [76]. For unilateral mating, equal concentrations (10⁷ cells/ml) of rav1Δ mutant cells of one mating type were mixed with wild-type cells of the opposite mating type. For bilateral mating, equal concentrations (10⁷ cells/ml) of rav1Δ mutant cells of both mating types were mixed. These mixtures were spotted onto a V8 mating medium (pH 5) and incubated in the dark at room temperature (25°C) for 7 to 25 days. Filamentous growth was monitored using a differential interference contrast (DIC) microscope (BX51; Olympus, Japan) and photographed using an Olympus BX51 microscope equipped with a SPOT Insight digital camera (Diagnostic Instruments, Inc.). For the cell fusion efficiency assay, each MATα and MATa strain bearing NAT or NEO markers, respectively, was mixed in an equal concentration (10⁷ cells/ml) and spotted (5 μl) onto the V8 medium. The cells were then incubated in the dark at room temperature for 24 or 48 h. After scraping and resuspending the cells, they were 100-fold diluted in distilled water, and 200 μl of each sample was spread onto a YPD medium containing both nourseothricin (100 μg/ml) and neomycin (50 μg/ml). After incubation in the dark at room temperature for 4 to 5 days, the number of colonies on each plate was counted using an automated bacterial colony counter (aCOLyte 3; Synbiosis Ltd., UK).

Total RNA preparation and quantitative RT-PCR

WT and RAV1 deleted strains were grown in 50 ml YPD broth at 30°C for 16 h. The cultures were then subcultured into 100 ml of fresh YPD broth and incubated until the OD₆₀₀ reached 0.8. At this point, the cells were harvested by centrifugation, frozen in liquid nitrogen for at least 30 min, and lyophilized. For stress response experiments, 50 ml of the culture was sampled as the zero time-point (basal condition), and the remaining 50 ml was treated with the indicated stress agents or conditions. Total RNAs were extracted as previously described [77], except using easy-Blue Total RNA extraction kit (iNtRON) instead of TRIzol extraction solution. The purified total RNA was then used for cDNA synthesis by reverse transcriptase (Thermo Scientific), and the resulting cDNA was used for quantitative PCR with specific primer pairs for each gene, using the CFX96 real-time system (Bio-Rad). Relative gene expression levels were monitored using the gene-specific primers listed in S5 Table.

Transmission electron microscopy

To prepare samples for electron microscopy analysis, WT and rav1Δ strains were cultured overnight at 30°C in the liquid YPD medium, and then sub-cultured to OD₆₀₀ of 0.8. One milliliter of cells was fixed for 12 h in 2% glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), washed in 0.1 M phosphate buffer, and post-fixed with 1% OsO₄ in 0.1 M phosphate buffer for 2 h. The cells were dehydrated using an ascending ethanol series (50, 60, 70, 80, 90, 95, 100%) for 10 min each and infiltrated with propylene oxide for 10 min. Specimens were then embedded with a Poly/Bed 812 kit (Polysciences) and polymerized in an electron microscope oven (TD-700, DOSAKA, Japan) at 65°C for 12 h. The block is equipped with a diamond knife in the Ultramichrome and is cut into 200 nm semi-thin sections, which were stained with toluidine blue for optical microscope observation. The region of interest was further cut into 80 nm thin sections using the ultramicrotome, placed on copper grids, double stained with 3% uranyl acetate for 30 min and 3% lead citrate for 7 min staining, and imaged with a transmission electron microscopy (JEM-1011, JEOL, Tokyo, Japan) at the acceleration voltage of 80 kV, equipped with a Megaview III CCD camera (Soft imaging system-Germany).

Insect-based in vivo virulence assay

For in vivo insect virulence assays, we used at least 15 Galleria mellonella caterpillars (Vanderhorst Wholesale, Inc., Saint Marys, OH, USA) in the final larval instar with a body weight of 200–300 mg, delivered within 7 days from the shipment date. Each wild-type H99, rav1Δ, and its complemented strain was incubated at 30 °C overnight, pelleted, washed three times with PBS, and resuspended in PBS at a 10⁶ cells/ml concentration. Four thousand C. neoformans cells per larvae were injected into the second-to-last prolegs of each larva using a 100-μl syringe equipped with a 10-μl needle and repeating dispenser (PB600-1, Hamilton Company, Reno, NV, USA). Negative control G. mellonella received PBS only. Infected larvae were placed in Petri dishes in a humidified container, incubated at 37 °C, and monitored daily. Larvae were considered dead when they turned black and showed no movement upon touching. Larvae that were pupated during the experiment were censored for statistical analysis. Prism 8 (GraphPad, San Diego, CA, USA) was used to illustrate survival curves, which were analyzed with a log-rank (Mantel–Cox) test.

Murine-based in vivo virulence assay

Animal experiments were conducted at the Core Facility Center for Zoonosis Research (Jeonbuk National University, South Korea). SPF/VAF-confirmed inbred 6-week-old female BALB/cAnNCrlOri mice were purchased from ORIENT BIO INC.(South Korea) and acclimatized to the breeding environment for one week before the experiment. For infection, strains were inoculated in fresh liquid YPD medium and cultured overnight a 30°C with shaking. The number of yeast cells was adjusted, and the mice were anesthetized with isoflurane before the strain was inhaled nasally. Mice were monitored daily for their condition, and survival rates were expressed as percent survival. For survival and histochemistry assay, 5 × 10⁵ cells/mouse was inhaled nasally, and monitored. Statistical analysis was performed using the log-rank (Mantel–Cox) test. For the fungal burden assay, 3 × 10⁶ cells/mouse was inhaled nasally, and lungs, liver, kidneys, spleen, and brain were collected on day 13 post-infection. Tissue weights were measured, and the colony-forming unit was determined by spreading the homogenized tissue on a YPD plate. The statistical significance of difference was determined using one-way ANOVA with Tukey’s multiple-comparison test. GraphPad Prism 9.5.1 was used for statistical analysis.

Histochemistry assay

For tissue staining, lungs were obtained from mice on day 14 after infection and fixed in 3.7% formalin solutions. After complete fixation, the tissue was dehydrated and clarified using xylene before being embedded in paraffin blocks. Tissue sections with a thickness of 5 μm were cut and stained with Grocott’s Methenamine Silver (GMS) Stain Kit (ab287884, abcam, UK), following the manufacturer’s protocol. The slide images were taken with a Primostar 3 (ZEISS, Germany) at a final 400× magnification.

In vitro BBB crossing assay

The BBB crossing assay was performed as previously described [78] with modifications using EndoGRO-MV complete media (Merck Millipore). Briefly, 5 × 10⁴ hCMEC/D3 cells in EndoGRO-MV medium supplemented with 1 ng/ml of FGF-2 were seeded on collagen (Corning) coated 8 μm-porous membranes (BD Falcon). The day after seeding, the medium was replaced with a fresh EndoGRO-MV medium supplemented with 2.5% human serum and grown for 4 days. A day before yeast inoculation, the medium was replaced with a new EndoGRO-MV medium supplemented with 1.25% human serum, and the cells were maintained at 37 °C and 5% CO₂ incubator. The integrity of tight junctions between hCMEC/D3 cells was confirmed by measuring the trans-endothelial electrical resistance (TEER), which should be around 200 Ω per cm². TEER was measured by Epithelial Volt per Ohm Metre (EVOM² device, World Precision Instruments). For BBB crossing assay, 5 × 10⁵ cells of C. neoformans WT (H99), rav1Δ, wdr1Δ, wdr1Δ rav1Δ mutants, and the rav1Δ::RAV1-mCherry strain were added to 100 μl of PBS and inoculated onto the top of the porous membranes. After 24 h incubation at 37 °C in a 5% CO₂ incubator, the number of yeast cells passing through the porous membrane was measured by counting CFU, and TEER was measured using the EVOM² device before and after inoculating yeast cells.

The BBB crossing ratio was determined using the following equation: the number of transcytosed CFU was divided by the total CFU (both transcytosed and non-transcytosed) for each tested strain. Subsequently, the ratio obtained for each tested strain was normalized to the corresponding value for the wild-type (WT) value.

Preparation for LC-MS/MS

Protein extraction methods were used in the previous study [79]. Rav1-mCherry strain was grown in 50 ml of YPD broth at 30°C for 16 h, subcultured into one litter of fresh YPD broth, and incubated until the culture reached an OD₆₀₀ of 0.8. At this point, the cells were harvested by centrifugation, frozen in liquid nitrogen for more than 30 min, and then lyophilized. To extract total proteins, the lyophilized cells were treated with lysis buffer (without SDS) containing 50 mM Tris-Cl (pH 7.5), 1% sodium deoxycholate, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 50 mM sodium fluoride (NaF), 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 2.5 × protease inhibitor cocktail solution (Merck Millipore). Total proteins were extracted as described previously [80]. The RFP-Trap Agarose (ChromoTek, USA) was added to the extracted whole cell lysates and incubated overnight while rotating at 4°C. Proteins bound to the agarose beads were washed three times and eluted with SDS sample buffer (50 mM Tris-Cl, 2% SDS, 10% glycerol, and 0.01% β-mercaptoethanol). Protein samples were loaded into 8% SDS-PAGE gel and run until the total band migration was about 1 cm. The gels were washed with distilled water and stained with the Coomassie Brilliant Blue G250 dye (CBB; Biosesang) overnight. The gel was washed three times with distilled water, and the CBB destaining buffer (10% methanol, 10% acetic acid, 80% distilled water) was added to remove any remaining coomassie blue buffers. The gel spots of interest for LC-MS/MS analysis were excised from the preparative gel and transferred into each 1.5 ml tube. The band was washed with 100 μL of distilled water; then, 100 μL of 50 mM NH₄HCO₃ (pH 7.8) and acetonitrile (6:4) were added to the band and shaken for 10 min. This process was repeated at least three times until the Coomassie brilliant blue G250 dye disappeared. The supernatant was decanted, and the band was dried in a speed vacuum concentrator (LaBoGeneAps, Lynge, Denmark) for 10 min. The solution was decanted and then digested with sequence-grade modified trypsin (Promega Co., Madison, WI, USA) (enzyme to substrate ratio = 1:50) at 37 °C with shaking for 16 h.

LC-MS/MS for peptides analysis

Nano LC-MS/MS analysis was performed with a Easy n-LC (Thermo Fisher San Jose, CA, USA). The capillary column used for LC-MS/MS analysis (150 mm × 0.075 mm) was obtained from Proxeon (Odense, Denmark) and the slurry packed in-house with a 5 μm, 100 A pore size Magic C18 stationary phase resin (Michrom BioResources, Auburn, CA). The mobile phase A for LC separation was 0.1% formic acid in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 3% B to 25% B in 70min, 25% B to 60% B in 13:59 min, 60% B to 95% B in 1:59 min, 95% B in 8:59 min, and 3% B in 6 min. The flow rate was maintained at 700 nl/min. LTQ-Orbitrap mass spectrometry (Thermo Fisher, San Jose, CA, USA) was used for the peptide identification. Mass spectra were acquired using data-dependent acquisition with a full mass scan (350–1200 m/z) followed by 10 MS/MS scans. For MS1 full scans, the orbitrap resolution was 15,000 and the AGC was 2×10⁵. For MS/MS in the LTQ, the AGC was 1×10⁴. The mascot server 2.6 (Matrixscience, USA) was used to identify peptide sequences present in a protein sequence database. Database search criteria were, database name; C. neoformans_H99_20210806 (15833 sequences; 8284938 residues), fixed modification; carbamidomethylated at cysteine residues; variable modification; oxidized at methionine residues, acetylated at N-term residue, deaminated at asparagine or glutamine residues, maximum allowed missed cleavage; 2, MS tolerance; 10 ppm, MS/MS tolerance; 0.8 Da. The peptides were filtered with a significance threshold of P<0.05. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [81] partner repository with the dataset identifier PXD042074 and 10.6019/PXD042074.