Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans

ABSTRACT The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target.

infections and 180,000 deaths globally each year (17), we first characterized the KEOPS subunit Bud32 as part of a global protein kinase functional survey (18). We focused on the atypical protein kinase due to its pleiotropic roles in C. neoformans. Bud32 is required but is not essential for the growth of C. neoformans and the deletion of BUD32 causes severe defects in the production of virulence factors (capsule, melanin, and urease), sexual differentiation, sterol biosynthesis, and pathogenicity (18). A Bud32 ortholog in fungal pathogens was also characterized in the filamentous fungus, Aspergillus fumigatus, wherein the ortholog, which was named PipA (PtkA-interacting protein A), is a binding partner for the cyclin-dependent kinase PtkA, a Cdk9 homolog, which functions in conidiophore development (19). Unlike the nonessential S. cerevisiae Bud32, PipA is essential in A. fumigatus (19), but its function has not been elucidated yet. In Fusarium graminearum, a plant fungal pathogen that causes head blight disease, the Bud32 ortholog, FgBud32, is required for aerial hyphal growth and branching, development of the conidium and perithecia, and pathogenesis (20). Nevertheless, the functions of the KEOPS complex in fungal pathogens have not been investigated mechanistically.
In this study, we comprehensively analyzed the pathobiological functions of the KEOPS complex components, Pcc1, Kae1, Bud32, and Cgi121, and its complex downstream signaling network in C. neoformans. We demonstrate that the KEOPS complex is not only important for growth and general stress response and adaptation but also critical for sterol biosynthesis, tRNA modification, transcriptional regulation, and pathogenicity of C. neoformans.
To analyze the function of the KEOPS complex in C. neoformans, we constructed two independent deletion mutants for the KAE1, PCC1, and CGI121 genes in the MATa C. neoformans H99 strain background (see Fig. S1D-F) and analyzed their phenotypic traits in comparison with those of the bud32D mutants. The bud32D, kae1D, and pcc1D mutants exhibited similar severe growth defects even at 30°C compared with the wildtype strain, whereas the cgi121D mutant showed moderate growth defects ( Fig. 1A and Fig. S2A-B). At the host physiological temperature (37°C), the pcc1D mutant showed the most evident growth defects, followed by kae1D, bud32D, and cgi121D mutants (Fig. 1A). Complementation with each mCherry-tagged wild-type allele successfully restored the normal growth in the KEOPS mutants (see Fig. S3). Supporting this, fluorescence-activated cell sorting (FACS) analysis of the wild-type and KEOPS mutant strains grown to the early logarithmic phase revealed that all the KEOPS complex mutants showed significantly decreased G 1 phase ( Fig. 1B and C; Fig. S2C). The pcc1D, kae1D, and bud32D mutants exhibited additional peaks indicating more genetic material than 2N ( Fig. 1B and C), suggesting that the CnKEOPS complex is involved in cell cycle control.
Cryptococcus neoformans has two mating types, MATa and MATa, and undergoes sexual differentiation to produce infectious basidiospores. When mated with the MATa wild-type strain (KN99a), MATa bud32D, kae1D, and pcc1D mutants did not generate any filamentous growth (Fig. 1D). In contrast, the cgi121D mutant produced intermediate levels of filamentation (Fig. 1D). Collectively, the CnKEOPS complex consists of Pcc1, Kae1, Bud32, and Cgi121 that play critical roles in the growth and development of C. neoformans.
The role of the KEOPS complex in stress responses and virulence of C. neoformans. Next, we addressed the role of the KEOPS complex in stress response and adaptation and production of two major virulence factors, melanin pigment and polysaccharide capsule, which contribute to the pathogenicity of C. neoformans. The bud32D, FIG 1 The roles of the KEOPS complex in the growth and sexual differentiation of C. neoformans. (A) The growth rates of wild type (WT) and mutants pcc1D (YSB4930), kae1D (YSB4863), bud32D (YSB1968), and cgi121D (YSB5037) were quantitatively measured at 30°C or 37°C using a multichannel bioreactor. (B, C) Cell cycle phases of propidium iodide (PI)-stained cells were quantitatively measured using flow cytometry. The flow cytometry graphs show representative data of three biological replicates. (B) The cell populations were represented by interphase (G 1 peak and S phase) and mitotic phase (G 2 /M peak). The proportion of cell populations from the same experiment was quantified and is shown in the graph. (C) One-way ANOVA was used to determine the statistically significant differences between the wild type and mutants. Error bars indicate SEM. (ns, not significant; *, P = 0.01-0.05, **, P = 0.001-0.01). (D) Filamentation and mating efficiency of the KEOPS mutants. The following MATa and MATa strains were cocultured on V8 medium (pH 5.0) for 10 days at room temperature in the dark: a (H99) Â a (KN99a), a pcc1D Â a, a kae1D Â a, a bud32D Â a, and a cgi121D Â a. One representative data from two independent experiments (see Fig. S2D  kae1D, and pcc1D mutants exhibited increased susceptibility to a phenylpyrrole class of fungicide fludioxonil, a reducing agent dithiothreitol, oxidative stress agents (diamide and tert-butyl hydroperoxide), genotoxic agents (methyl methanesulfonate and hydroxyurea), and osmotic stresses (NaCl, KCl, sorbitol) but displayed increased resistance to fluconazole and an ER stress agent tunicamycin ( Fig. 2A-B; Fig. S3 and S4A). All Each strain was cultured overnight in YPD broth at 30°C, serially diluted 10-fold, and then spotted onto YPD medium containing the following chemicals: 1 mg/mL fludioxonil (FDX), 16 mg/mL fluconazole (FCZ), 0.02 mM menadione (MD), 100 mM hydroxyurea (HU), 1 M NaCl, or 4 mg/mL tunicamycin (TM). The plates were incubated at 30°C for 4 to 6 days. (C) Melanin production assay. Indicated strains were spotted onto Niger seed, L-DOPA, and epinephrine agar medium containing 0.1% or 0.2% glucose, incubated at 37°C, and photographed after 1 to 2 days. hog1D and cac1D were used as control strains and showed enhanced and reduced melanin production, respectively. Representative images from three independent experiments are shown here. (D) Capsule production assay. Indicated strains were cultured in capsule-inducing media (Fetal bovine serum [FBS] and Littman's [LIT] medium) at 37°C for 2 days. cac1D is an acapsular mutant. The cells were scraped and observed using India ink staining. Capsule thickness (total diameter 2 cell body diameter) was measured for 50 WT and mutant cells (n = 50). Statistical significance was calculated using one-way ANOVA analysis with Bonferroni's multiple-comparison test. Data are presented as mean values 6 SEM (****, P , 0.0001). One representative graph is shown. (E) Insect-killing assay for measuring the virulence of KEOPS mutants. P values were calculated using the log-rank (Mantel-Cox) test to measure statistical differences between WT strain (H99S) and each mutant strain.
Roles of the Cryptococcal KEOPS Complex mBio three mutants also exhibited highly reduced production of melanin ( Fig. 2C; see Fig. S4B) and capsule ( Fig. 2D; Fig. S4C). In contrast, the cgi121D mutant exhibited generally similar but milder phenotypic alterations than the other KEOPS mutants ( Fig. 2A-D). Collectively, these results indicate that Pcc1, Bud32, and Kae1 play major roles in stress and antifungal drug responses and virulence factor production in C. neoformans, whereas Cgi121 has less pronounced but still evident roles. Next, we examined the role of KEOPS components in the virulence of C. neoformans using the insect-killing assay. The kae1D and pcc1D mutants exhibited highly reduced virulence like the bud32D mutant (18) (Fig. 2E). Notably, the cgi121D mutants, which exhibited intermediate in vitro virulence-related phenotypes compared with other KEOPS mutants, also showed highly reduced virulence (Fig. 2E). Collectively, these results indicate that all KEOPS components are critical for the pathogenicity of C. neoformans.
The fact that Bud32, Kae1, Pcc1, and Cgi121 play redundant functions suggests that the four components may form a complex in C. neoformans. In S. cerevisiae, Gon7-Pcc1-Kae1-Bud32-Cgi121 has a linear arrangement (10,22). To verify the linear arrangement of the KEOPS components in C. neoformans, first we examined whether Pcc1 and Kae1 interact with each other using a coimmunoprecipitation (co-IP) experiment. We constructed the KAE1:KAE1-6xHA, where the 6xHA was tagged at the C terminus of Kae1 (see Fig. S5E). Subsequently, we tagged the C-terminal region of the PCC1 allele with 4xFLAG in both wild-type and the KAE1:KAE1-6xHA strains (see Fig. S5F). Chromosomal tagging of Pcc1 and Kae1 with 4xFLAG and 6xHA, respectively, did not cause growth defects in C. neoformans (Fig. S5G), indicating that these alleles were functional. Co-IP analysis revealed that Pcc1 physically interacted with Kae1 in C. neoformans (Fig. 3B). We next examined whether Bud32 interacts with Kae1 in C. neoformans. We constructed the bud32D::BUD32:4xFLAG strain, where the 4xFLAG was tagged at the C terminus of Bud32 (Fig. S5H). Reintegration of the BUD32:4xFLAG allele successfully restored normal phenotypes in the bud32D mutant (Fig. S5G), indicating that the Bud32-4xFLAG allele was functional. Subsequently, we tagged the C-terminal region of the KAE1 allele with 6xHA in the bud32D::BUD32:4xFLAG strain (Fig. S5E). Co-IP analysis revealed that Kae1 physically interacted with Bud32 in C. neoformans (Fig. 3C).
Lastly, we addressed whether Bud32 interacts with Cgi121 by tagging the N-or C-terminal region of the CGI121 allele with 6xHA in the wild-type and bud32D::BUD32:4xFLAG strains (Fig. S5I). The N-terminal chromosomal tagging of Cgi121 with 6xHA (6xHA-Cgi121) did not restore normal phenotypes in the cgi121D mutant (Fig. S5J), indicating that 6xHA-Cgi121 was not functional. In contrast, the C-terminal chromosomal tagging of Cgi121 with 6xHA (Cgi121-6xHA) restored normal phenotypes in the cgi121D mutant (Fig. S5J), indicating that Cgi121-6xHA was functional. However, cells having both Bud32-4xFLAG and Cgi121-6xHA alleles exhibited minor growth defects and increased stress susceptibility (Fig. S5J), implying that these two tagged Bud32 and Cgi121 proteins may have a problem in normal interaction. We did not find any interaction between Bud32 and Cgi121 through co-IP analysis (data not shown). Altogether, the KEOPS complex in C. neoformans appears to be composed of a linear arrangement of Pcc1-Kae1-Bud32, and Cgi121 may transiently bind, if at all, to Bud32.
The essential role of the extended loop region of Bud32 for KEOPS complex functionality in C. neoformans. Based on physical interaction data between Pcc1, Kae1, and Bud32, we attempted to predict the CnKEOPS complex structure using AlphaFold2 multimer (23). The CnKEOPS complex appears to have a linear arrangement in the order Pcc1-Kae1-Bud32-Cgi1 that is highly similar to that of the yeast KEOPS complex (22) (Fig. 4A). However, comparing cryptococcal and yeast KEOPS complex structures reveals that CnBud32 contains an internal extended loop structure (Loop 3) not observed in the yeast Bud32 (Fig. 4A). The loop 3 is composed of 59 amino acids (V114-M172), including 16 glutamate residues conferring it a high negative charge. To examine whether the extended loop 3 in CnBud32 is important for Bud32 function, we replaced it with the corresponding short loop 3 region of yeast Bud32 (BUD32 ScL3 ) and constructed the bud32D::BUD32 ScL3 strain using complementation ( Fig. 4B; Fig. S6A-B). Notably, reintegration of the BUD32 ScL3 allele failed to restore the phenotypes of the bud32D mutant (Fig. 4C), indicating that the extended loop 3 region is critical for the function of CnBud32. Collectively, cryptococcal Bud32 has a structurally unique feature compared with yeast Bud32.
Kinase activity-dependent and -independent roles of Bud32 in C. neoformans. Next, we checked whether the kinase activity of Bud32 is required for the function of the KEOPS complex in C. neoformans. We first examined whether Bud32 has kinase activity at all in C. neoformans. For this purpose, we expressed recombinant Bud32 protein in Escherichia coli (see Fig. S6C). After expressing and purifying Bud32 protein, we measured its kinase activity. Interestingly, we found that Bud32 undergoes autophosphorylation ( Fig. 5A and B). The presence of a kinase substrate (a-casein) did not increase kinase activity in Bud32 (Fig. 5B).
We then questioned whether the kinase activity is essential for the pleiotropic function of Bud32. We constructed the kinase-dead allele of BUD32 (BUD32 K54A ) through the site-directed mutation of K54A that is required for ATP binding (equivalent to S. cerevisiae K52 residue) and reintegrated it into the native BUD32 locus (Fig. S6D). Surprisingly, reintegration of the BUD32 K54A allele completely restored the growth of C. neoformans at 30°C, amphotericin B resistance, and mating efficiency like the wild-type BUD32 allele (Fig. 5C-E). In contrast, the bud32D::BUD32 K54A complemented strain had partially restored resistance to hydrogen peroxide and high temperature (39°C) and melanin and capsule production (Fig. 5F). However, the reintegration of the BUD32 K54A allele failed to recover the susceptibility to fluconazole and tunicamycin (Fig. 5C).
As the kinase activity differentially contributed to the functions of Bud32, we checked whether Bud32 kinase activity is required for the virulence of C. neoformans using a murine model of systemic cryptococcosis (Fig. 5G). As expected from the previous signature-tagged mutagenesis (STM)-based murine infectivity and insect-killing assays (18), the bud32D mutant was avirulent upon intranasal infection of mice (Fig. 5G). In contrast, the bud32D::BUD32 complemented strain was as virulent as the wild-type H99 strain, but the bud32D::BUD32 K54A complemented strain showed an intermediate level of virulence (Fig. 5G), indicating that the Bud32 kinase activity partially contributes to the virulence of C. neoformans. Collectively, all these results suggest that Bud32 plays pleiotropic roles in C. neoformans in both kinase activity-dependent and -independent manners.
The role of the KEOPS complex in the tRNA modification of C. neoformans. Next, we addressed whether the CnKEOPS complex plays an evolutionarily conserved role in tRNA modification by catalyzing the t 6 A modification of tRNAs (summarized in Fig. 6A). For this purpose, we performed a primer extension analysis of a t 6 A-containing tRNA (Ile AAU) isolated from wild type, and bud32D, bud32D::BUD32, bud32D::BUD32 K54A , pcc1D, kae1D, and cgi121D mutants. The primer extension profile of the bud32D mutant was markedly different at position 39, equivalent to position 37 of yeast tRNA (Ile AAU), from that of the wild type and bud32D::BUD32 complemented strain ( Fig. 6B; Fig. S7A). The primer extension profiles of the pcc1D, kae1D, and cgi121D mutants were similar to those of the bud32D mutant ( Fig. 6B;  Fig. S7A). The bud32D::BUD32 K54A strain exhibited an intermediate primer extension profile between the wild type and bud32D mutant ( Fig. 6B; Fig. S7A), suggesting that the kinase activity of Bud32 is required, but not essential, for the t 6 A modification. When we performed the primer extension analysis of a non-ANN codon-recognizing tRNA (Val The tetra-heterocomplex structure of C. neoformans Pcc1, Kae1, Bud32, and Cgi121 was predicted using AlphaFold2 multimer. Query sequence was ordered, and the MSA option and Advanced settings were used as default. The extended loop 3 region, characteristic of cryptococcal Bud32, is marked with a green circle. (B) The unique extended loop 3 region of cryptococcal Bud32 was replaced with the short loop 3 region of S. cerevisiae. The yeast Bud32 sequence of L113 to T140 was introduced between S114 and H172 of cryptococcal Bud32. (C) Each strain (WT, bud32D [YSB1968], bud32D::BUD32 [YSB4662], and bud32D::BUD32 Loop3 [YSB6211 and YSB6209]) was cultured overnight in liquid YPD medium at 30°C, serially diluted 10-fold, spotted onto YPD medium containing 0.8 mg/mL amphotericin (AMB), 22 mg/mL FCZ, 0.4 mg/mL TM, 0.03 mM MD, 120 mM HU, 5 mg/mL CFW, 0.02% sodium dodecyl sulfate (SDS), 1.5 M KCl, or 1.5 M NaCl, and incubated at 30°C for 3 days. For the high-temperature sensitivity assay, 10-fold serially diluted cells were spotted on YPD medium and then incubated at 39°C for 3 days. (Continued on next page) AAC) isolated from the same set of strains, all KEOPS mutant strains exhibited very similar primer extension profiles ( Fig. 6C and S7B). All these results indicate that the CnKEOPS components play a conserved role in t 6 A modification of ANN codon-recognizing tRNAs.

Roles of the Cryptococcal KEOPS Complex mBio
The role of the cryptococcal KEOPS complex as a transcriptional regulator. The yeast KEOPS complex also serves as a transcription factor by interacting with chromatin in vivo and can regulate gene expression by recruiting transcriptional coactivators such as SAGA and Mediator (3). To investigate the roles of the CnKEOPS complex as a transcription regulator, we analyzed the transcriptome profiles of wild-type, bud32D, bud32D::BUD32 K54A , and kae1D mutants using RNA-sequencing (RNA-seq) (Fig. 7). The principal-component analysis of the RNA-seq data revealed that the transcriptome profiles of bud32D and kae1D mutants were clustered together, but distinct from those of wild type and bud32D::BUD32 K54A mutant (see Fig. S8A). Deletion of BUD32 significantly altered the basal expression levels of 431 genes (.2-fold cutoff; P , 0.05; 207 downregulated and 224 upregulated genes) (Fig. 7A). Similarly, deletion of KAE1 significantly changed the basal expression levels of 298 genes (.2-fold cutoff; P , 0.05; 110 downregulated and 188 upregulated genes) (Fig. 7B). Notably, the transcriptome profile of the bud32D mutant was highly similar to that of the kae1D mutant ( Fig. 7C; Fig. S8B and Data Set S2), indicating that Bud32 and Kae1 constitutes a transcriptional regulator complex. In contrast, a lack of the BUD32 kinase activity affected only a small number of genes (2 downregulated and 10 upregulated genes) (Fig. 7D), suggesting that the Bud32 kinase activity is not critical for the transcriptional regulator activity.
In agreement with our finding that the KEOPS complex is critical for the cryptococcal cell growth (Fig. 1B), differentially expressed gene (DEG) analysis revealed that many Bud32-and Kae1-dependent genes were related to carbon and nitrogen metabolism, amino acid metabolism, and transporter activity ( Fig. 7E and F). Bud32 and Kae1 appeared to regulate proline and leucine metabolism genes, such as PUT1, PUT5, LEU2, and amidase gene (CNAG_01072). Correspondingly, the expression of several transporter genes appeared to be regulated by both Bud32 and Kae1, which might be a compensatory response to the altered nutrient utilization in the KEOPS mutants. Most notably, the deletion of BUD32 or KAE1 downregulated ergosterol biosynthesis-related genes, such as ERG130, ERG1, and ERG6 (Data Set S2). The transcription factors regulated by Kae1 and Bud32 include STB4 and MLN2. Supporting the role of the KEOPS complex in the sexual reproduction of C. neoformans, several pheromones and matingrelated genes (CNAG_07406, CNAG_06845, and ZIP1) were commonly upregulated by Bud32 and Kae1. Taken together, the KEOPS complex serves as a major transcriptional regulator in C. neoformans and regulates genes involved in carbon and nitrogen metabolism, amino acid metabolism, sterol biosynthesis, pheromone response and mating, and transcription factor regulation.
The role of cryptococcal KEOPS complex as a major regulator of sterol biosynthesis-related genes. The transcriptome analysis revealed that several ergosterol biosynthesis-related genes were upregulated by Bud32 and Kae1, suggesting that the CnKEOPS complex plays a central role in ergosterol biosynthesis-related gene expres- ) was cultured, diluted, and spotted as described in Fig. 2A onto YPD medium containing 1.4 mg/mL AMB, 22 mg/mL fluconazole, 0.5 mg/mL TM, or 0.02 mM MD, and then incubated at 30°C for 3 days. For the high-temperature sensitivity assay, 10-fold serially diluted cells were spotted on YPD medium and then incubated at 39°C for 3 days. (D) The growth rate of each indicated strain was quantitatively measured at 30°C for 50 h using a multichannel bioreactor. One representative graph from two independent experiments is shown here. (E) Mating assay. The following MATa and MATa strains were cocultured on V8 medium (pH 5.0) for 10 days at room temperature in the dark: a (H99) Â a (KN99a), a bud32D Â a, a bud32D::BUD32 Â a, and a bud32D:: BUD32 K54A Â a. (F) The role of the Bud32 kinase activity in melanin and capsule production. The melanin pigments in each strain were induced at 37°C using Niger seed or L-DOPA medium containing 0.1% or 0.2% glucose, respectively, and photographed after 2 to 3 days. For capsule production assay, each strain was spotted on FBS agar medium and incubated at 37°C for 2 days. Statistical significance was calculated using one-way ANOVA analysis with Bonferroni's multiple-comparison test. Data are presented as mean values 6 SEM (****, P , 0.0001; NS, not significant). (G) The role of the Bud32 kinase activity in C. neoformans virulence. The survival rates of mice intranasally infected with the indicated strains were monitored for 63 days. Seven mice per strain were used.
Roles of the Cryptococcal KEOPS Complex mBio sion. Therefore, we investigated the regulatory role of the KEOPS complex in ergosterol biosynthesis-related gene expression in response to azole treatment. Fluconazole treatment generally increased the induction of ergosterol biosynthesis-related genes, such as ERG2, ERG4, ERG6, ERG7, ERG11, and ERG25 in the wild-type strain but not in the bud32D mutant (Fig. 8A). Reintegration of BUD32 restored the normal induction of the ERG genes in the bud32D mutant (Fig. 8A). Therefore, it is evident that Bud32 is critical for regulation of sterol biosynthesis-related genes. Similarly, deletion of PCC1 and KAE1 abolished the induction of these genes under sterol depletion by fluconazole treatment, whereas deletion of CGI121 partially reduced their induction (Fig. 8B).
Notably, the kinase-dead allele of BUD32 (BUD32 K54A ) also restored the normal induction of the ERG genes in the bud32D mutant (Fig. 8C), indicating that the kinase activity of Bud32 is unessential for the regulation of sterol biosynthesis-related genes.
Despite the lack of azole-mediated induction of ergosterol biosynthesis-related genes in the KEOPS mutants, they showed increased resistance to fluconazole ( Fig. 2A). Induction of ergosterol biosynthesis-related genes upon sterol depletion by fluconazole treatment is a compensatory mechanism to maintain intracellular sterol homeostasis. Therefore, we hypothesized that deletion of the KEOPS complex could inhibit sterol depletion by azole treatment, resulting in incomplete induction of ergosterol biosynthesis-related genes by fluconazole treatment. To address this possibility, we measured intracellular ergosterol contents in the wild type and KEOPS mutant strains with or without fluconazole treatment. Fluconazole treatment significantly reduced the ergosterol content in the wild-type strain but not in the KEOPS mutants, supporting our hypothesis (Fig. 8D). Taken together, the KEOPS complex is involved in the regulation of fungal sterol content and subsequent ergosterol biosynthesis-related gene expression.

DISCUSSION
In this study, we identified and functionally characterized the fungal KEOPS complex, consisting of Pcc1, Kae1, Bud32, and Cgi121 in C. neoformans. This evolutionarily conserved protein complex has been structurally and functionally characterized in the model budding yeast S. cerevisiae but not in fungal pathogens. An ortholog of Gon7, an additional subunit of the yeast KEOPS complex, has not been identified in C. neoformans. Based on our bioinformatics analysis, several fungal species do not contain Gon7 orthologs, suggesting that Gon7 is not an evolutionarily conserved KEOPS component at least in fungi. In humans, C14ORF142 has been recently identified as the ortholog of yeast Gon7 (24). Here, we demonstrate that the KEOPS complex plays pivotal roles in growth and cell cycle control, differentiation, stress responses, tRNA modification, transcriptional regulation, and sterol biosynthesis in C. neoformans. Therefore, disruption of any one of KEOPS components significantly impacts the virulence of C. neoformans (summarized in Fig. 9).
A previous structural and functional analysis of the yeast KEOPS complex revealed that it consists of a linear arrangement of Gon7-Pcc1-Kae1-Bud32-Cgi121 (10,22). Supporting this, we also observed physical interactions between each pair of the CnKEOPS components: Pcc1-Kae1 and Kae1-Bud32. The CnKEOPS complex structure predicted using AlphaFold2 is also like the yeast Pcc1-Kae1-Bud32-Cgi121 structure. Among these components, Pcc1, Kae1, and Bud32 appeared to be the central components of the CnKEOPS complex, whereas Cgi121 likely serves as an auxiliary component because the pcc1D, kae1D, and bud32D mutant phenotypes were indistinguishable, but the cgi121D mutant exhibited intermediate phenotypes. This is in good agreement with the previous finding that Pcc1, Kae1, and Bud32 are required for the archaeal KEOPS function, but Cgi121 only increases its efficiency (25). In several archaea, Kae1 and Bud32 are often fused as a single protein (26,27). However, one notable difference we observed is the presence of the extended internal loop 3 of Bud32 in C. neoformans. We found that the extended internal loop 3 is critical for the function of Bud32. According to the predicted complex structure, the loop 3 region of C. neoformans is adjacent to the Kae1 protein, suggesting the possible interaction between them; however, the region would be flexible and has very low model confidence according to the structure prediction result (the IDDT score is less than 30). It was previously reported that Bud32 in S. cerevisiae shows kinase activity after forming a functional unit with Kae1, which has ATPase activity (22,25). The fact that CnBud32 is a kinase with a phosphotransferase domain, and Kae1 has a substrate binding site and an ATPase domain supports the possibility that the two proteins act as a unit in C. neoformans.
The yeast KEOPS complex has three major biological functions: (i) tRNA modification, (ii) transcriptional regulation, and (iii) telomere regulation. We also found that the first two roles are conserved in the CnKEOPS complex. All four KEOPS components, Pcc1, Kae1, Bud32, and Cgi121, were involved in catalyzing t 6 A modification at position 37 of ANN codon-recognizing tRNAs, such as Ile AAU. We found that the kinase activity of Bud32 is required, but not essential, for the t 6 A modification in C. neoformans, consistent with the finding in S. cerevisiae where the kinase activity of Bud32 is partially required for the t 6 A modification (28). In addition, KEOPS evidently works as a transcriptional regulator in C. neoformans. Our RNA-seq-based transcriptomic analysis revealed that deletion of BUD32 and KAE1 led to altered expression of several hundreds of genes, particularly those involved in carbon and nitrogen metabolism, amino acid metabolism, pheromone response/mating, and transport. The previous DNA microarray-based transcriptome analysis in S. cerevisiae also reported that genes involved in Roles of the Cryptococcal KEOPS Complex mBio carbohydrate and amino acid biosynthesis and pheromone response are regulated by the yeast KEOPS complex (3,29), suggesting that these KEOPS functions are conserved in fungi.
Despite the conserved roles of the CnKEOPS complex in tRNA modification and transcriptional regulation, its role in telomere regulation remains unclear. In S. cerevisiae, the KEOPS complex regulates telomerase recruitment or telomerase activity at double-strand breaks (2). Deleting the KEOPS complex leads to short telomeres and prevents the addition of new telomeres to DNA double-strand breaks (2). However, when we monitored the subtelomeric regions of chromosomes 1, 4, and 5 of C. neoformans using Southern blot analysis, we observed no difference in telomere length between the wild type and KEOPS mutant strains (data not shown). The recent RNAseq analysis revealed that the genes in the subtelomeric region are upregulated in the S. cerevisiae KEOPS mutants (30). However, based on our RNA-seq data, we could not

Roles of the Cryptococcal KEOPS Complex mBio
find any significant changes in the expression level of genes in the subtelomeric region of KEOPS mutants. Therefore, the potential role of the CnKEOPS complex in telomere silencing should be further investigated. Besides the conserved roles, this is the first study to report the role of the fungal KEOPS complex in the regulation of sterol biosynthesis-related genes. Fluconazole-mediated induction of ERG2, ERG7, ERG11, and ERG25 expression was completely dependent on Pcc1, Kae1, and Bud32 but only partially on Cgi121. Notably, the kinase activity of Bud32 appeared to be dispensable for this function. However, our data implied that the CnKEOPS complex indirectly affects ergosterol biosynthesis-related gene expression by affecting cellular sterol content. If the KEOPS complex is directly involved in the induction of ergosterol biosynthesis-related genes, sterol contents upon azole treatment should be lower in the KEOPS mutants than in the wild-type strain. However, we found that cellular sterol contents in the KEOPS mutants were maintained even after fluconazole treatment, possibly explaining the greater fluconazole resistance of KEOPS mutants than the wild-type strain. KEOPS complex might regulate genes involved in azole uptake. Supporting this, our RNAseq analysis showed that several transporter and transmembrane protein genes are regulated by the KEOPS complex. Whether such transporters are involved in fluconazole uptake remains to be investigated.
Our data clearly demonstrated that the fungal KEOPS complex is critical for the pathogenicity of C. neoformans, which is not surprising considering its critical role in the growth and virulence factor production of C. neoformans. Interestingly, the kinasedead BUD32 K54A strain exhibited normal growth and significantly restored virulence factor production but with highly attenuated virulence. These data suggest that the kinase activity of Bud32 is also required for fungal pathogenicity. Supporting this, Bud32 is known to phosphorylate and/or interact with other substrates apart from the KEOPS complex (7,9,31). In S. cerevisiae, Bud32 phosphorylates Ser134 of the monothiol glutaredoxin Grx4p and is itself regulated by the Sch9 kinase (32). Grx4 interacts with Cir1, a key regulator for iron uptake and virulence of C. neoformans and essential for its pathogenicity (33). However, the functional and mechanistic connection between Sch9, Bud32, and Grx4 is not clear in C. neoformans due to the following reasons. First, although the grx4D mutants exhibit severe defects in growth at 30°C and 37°C and the production of capsule and melanin (33), our result demonstrated that the kinase-dead BUD32 K54A strain showed normal growth and significantly restored virulence factor production. Therefore, other Bud32 substrates could be responsible for the attenuated virulence of the kinase-dead BUD32 K54A strain. Second, although the Ser258 residue of the yeast Bud32 is phosphorylated by Sch9 (32), the corresponding residue is not conserved in CnBud32. Therefore, the KEOPS-independent Bud32 substrates and the potential Sch9-Bud32-Grx3 signaling cascade in C. neoformans remain to be elucidated. However, we also cannot exclude the possibility that the BUD32 K54A allele may confer some residual kinase activity at high concentrations of ATP, thus partly restoring certain phenotypes of the bud32D mutant in C. neoformans.
There is a compelling possibility that the KEOPS complex could be exploited as potential antifungal drug targets. The fungal KEOPS components could be targeted by inhibiting their enzymatic activity (e.g., the kinase activity of Bud32), structural integrity, or protein-protein interactions. In C. neoformans, for example, our data suggest that perturbation of the KEOPS complex could not only reduce the viability and virulence of the pathogen within the host, but also synergize with the antifungal activity of amphotericin B, which is widely used for the treatment of cryptococcal infection. However, it will be challenging to specifically target the fungal KEOPS complex, but not the human KEOPS complex, due to their structural and functional similarities. In the case of cryptococcal infections, the unique but functionally critical, extended loop of CnBud32 could be targeted for developing anticryptococcal-specific drugs. Based on these findings, protein structure-based chemical design and development targeting the fungal KEOPS complex is warranted in future studies.

MATERIALS AND METHODS
Ethics statement. Animal care and all experiments were conducted in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (NTU). The NTU IACUC approved all the vertebrate studies (NTU107-EL-00111).
Construction of KEOPS mutant strains. The KEOPS complex mutant strains were constructed in the C. neoformans serotype A H99S strain background. Gene disruption cassettes containing the nourseothricin-resistance marker (nourseothricin acetyltransferase; NAT) with indicated signature-tagged sequences were generated using conventional overlap PCR or NAT split marker/double-joint (DJ) PCR strategies, as previously reported (34,35). For gene deletion of KAE1, PCC1, and CGI121, NAT signaturetagged sequences (numbers 212, 43, and 184) were used in gene disruption cassettes, respectively. All primers used in this study are listed in Data Set S1. In the first round of PCR, the 59-and 39-flanking regions for the targeted genes (KAE1, PCC1, and CGI121) were amplified with primer pairs L1/L2 and R1/ R2, respectively, using H99S genomic DNA as a template. For the overlap PCR, the whole NAT marker was amplified with the primers M13Fe (M13 forward extended) and M13Re (M13 reverse extended) using a pNAT-STM plasmid containing the NAT gene with each unique signature-tagged sequence. For the split marker/DJ-PCR, the split 59-and 39-regions of the NAT marker were amplified with primer pairs M13Fe/SM2 and M13Re/SM1, respectively, with the plasmid pNAT-STM numbers 212, 43, or 184. In the second round of overlap PCR, the gene disruption cassettes were amplified with primers L1 and R2 by combining the first round PCR products as the templates. In the second round of split marker/DJ-PCR, the 59-and 39-regions of NAT-split gene disruption cassettes were amplified with primer pairs L1/SM2 and R2/SM1, respectively, by combining the corresponding first-round PCR products as the templates. For biolistic transformation, the H99S strain was cultured overnight at 30°C in 50 mL yeast extract-peptone-dextrose (YPD) medium, pelleted, and resuspended in 5 mL of distilled water. Approximately 200 mL of the cell suspension was spread on YPD solid medium containing 1 M sorbitol and further incubated at 30°C for 3 h. The PCR-amplified gene disruption cassettes were coated onto 600 mg of 0.6 mm gold microcarrier beads (Bio-Rad) and biolistically introduced into the cells using a particle delivery system (PDS-100, Bio-Rad). The transformed cells were further incubated at 30°C to recover cell membrane integrity, scraped after 4 h, and transferred to the selection medium (YPD solid plate containing 100 mg mL 21 nourseothricin; YPD1NAT). Stable NAT-positive transformants were selected through more than two passages on the YPD1NAT plates. All NAT-positive strains were first screened using diagnostic PCR with each screening primer listed in Data Set S1. To verify accurate gene deletion and the absence of any ectopic integration of each gene disruption cassette, Southern blot analysis was performed for all the mutants (Fig. S1D-F). To validate a mutant phenotype and exclude any unlinked mutational effects, we constructed more than two independent deletion strains for each mutant.
To construct strains containing KAE1-4xFLAG or PCC1-6xHA, each cassette for C-terminal chromosomal tagging was generated using a split marker/double-joint PCR strategy, as previously reported (34,35). The 39-ORF regions, 39-flanking regions of each gene, and plasmids containing the 4xFLAG or 6xHA sequence were separately amplified in the first round of PCR. Then the 39-ORF region and NAT-spilt marker for KAE1-4xFLAG or the hygromycin B resistance gene (HYG; hygromycin B phosphotransferase) split marker for PCC1-6xHA were combined. Also, the 39-flanking region and each dominant selectable split marker were combined in the second round of PCR. The tagging cassettes were delivered into the H99S strains or each tagged strain through biolistic transformation. The genotypes of tagged strains were confirmed using Southern blot.
Construction of Bud32 kinase-dead mutant and Bud32 containing the short loop region of yeast Bud32. To construct the kinase-dead BUD32 (BUD32 K54A ) strain, site-directed mutagenesis was conducted as previously described (37). The plasmid containing the BUD32 allele was amplified with mutagenic primers listed in Data Set S1. The products were digested with DpnI for 2 to 3 h at 37°C. After the reaction, the purified DNA was transformed into E. coli. The plasmid containing the BUD32 K54A allele was confirmed through sequencing and subcloned into pNEO to generate pNEO_BUD32 K54A . Then, the ClaI-digested linearized pNEO_BUD32 K54A was integrated into the BUD32 native locus. To construct the bud32D::BUD32 ScL3 strain where the extended loop 3 region of CnBud32 was replaced with the short loop region of yeast Bud32, the DNA fragments spanning the promoter and loop3 upstream regions, the loop3 downstream and terminator regions, and the short loop region of yeast Bud32 were amplified Roles of the Cryptococcal KEOPS Complex mBio using PCR with primers listed in Data Set S1. The first and second PCR products were combined and used as the templates for overlap PCR using primers B7763 and B9316. The second and third PCR products were combined and used as the templates for overlap PCR using primers B9313 and B7764. Finally, two overlap PCR products were combined and used for the third round of PCR with primers B9366 and B9367. The final DNA product was subcloned into pNEO plasmid (pNEO_BUD32 ScL3 ), digested with ClaI, and introduced into the bud32D mutants (YSB1968). Imaging cellular localization of mCherry-tagged proteins. The mCherry-tagged strains were incubated in YPD broth overnight at 30°C and subcultured in fresh YPD liquid media until their optical density at 600 nm (OD 600 ) reached 0.8. The cells were fixed with 4% paraformaldehyde containing 3.4% sucrose for 15 min at room temperature. After incubation, the cells were pelleted, washed with 0.1 M potassium phosphate buffer (pH 7.5) containing 1.2 M sorbitol, and then stored in potassium phosphate buffer. For nuclear staining, the cells were incubated with 10 mg/mL of Hoechst 33342 (Thermo Fisher) in the dark for 30 min. After incubation, samples were observed and photographed using differential interference contrast (DIC) and fluorescence microscopy (Nikon Eclipse, Tokyo, Japan) equipped with a digital camera (DS-Qi2).
Coimmunoprecipitation and Western blot. 4xFLAG-and/or 6xHA-tagged strains were incubated in YPD liquid medium overnight at 30°C, subcultured in fresh YPD broth, further incubated until OD 600 = 0.8, pelleted, and frozen in liquid nitrogen. The frozen cell pellet was diluted with lysis buffer containing 50 mM Tris-Cl (pH 7.5), 1% sodium deoxycholate, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 50 mM NaF, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and 1Â protease inhibitor cocktail solution (Calbiochem, USA). The whole-cell lysates were obtained after bead beating. Anti-FLAG antibody (Sigma-Aldrich, F1804) was added to the lysates and incubated overnight at 4°C rotating. Then, Sepharose protein G beads (GE Healthcare Life Sciences) were added, and the mixture was incubated for 6 h at 4°C rotating. The collected Sepharose beads were washed thrice with lysis buffer containing 50 mM NaCl. The protein bound to the beads was eluted with SDS sample buffer (50 mM Tris-Cl, 2% SDS, 10% glycerol, and 0.01% b-mercaptoethanol) and detected with anti-FLAG (Sigma-Aldrich, F1804) and anti-HA (Santa Cruz Biotechnology, sc-7392) antibodies.
Growth and chemical susceptibility test. Each strain was grown in YPD broth for 16 h at 30°C, serially diluted 10-fold (dilution factor: 1 to 10 4 ), and spotted onto YPD solid medium containing the indicated concentrations of chemical agents to induce environmental stresses. Cells were incubated at 30°C for 1 to 5 days and photographed daily. To analyze the growth rate of KEOPS mutants, the wild-type strain (H99S) and mutants were incubated at 30°C overnight and their cell concentrations were adjusted to OD 600 = 0.2 in fresh YPD liquid medium. Cells were then incubated at 30°C in a multichannel bioreactor (Biosan Laboratories, Inc., Warren, MI, USA), and OD 600 was automatically measured for 70 h.
Mating. To monitor unilateral mating efficiency, each MATa mutant and wild-type (H99S) strain was mated with the MATa KN99a strain. Each cell was cultured in YPD broth for 16 h, pelleted, and washed twice with phosphate-buffered saline (PBS). The MATa cells (10 7 cells/mL) were mixed with the equal volume (10 7 cells/mL) of MATa KN99a cells, spotted on V8 mating media (pH 5), and incubated in the dark for 10 days. Filamentous growth was observed and photographed using DIC microscopy (BX51, Olympus) equipped with a SPOT Insight digital camera (Diagnostic Instrument Inc.).
Virulence factor production assay. For the melanin production assay, cells cultured overnight were washed twice with PBS and spotted on Niger seed, dopamine, or epinephrine agar media (1 g L-asparagine, 3 g KH 2 PO 4 , 250 mg MgSO 4 , 1 mg thiamine, 5 mg biotin, and 100 mg L-DOPA or epinephrine hydrochloride per L) containing 0.1% or 0.2% glucose. The cells were incubated at 37°C and photographed for 1 to 3 days. The capsule production assay was performed using two types of capsule-inducing media: FBS (10% of fetal bovine serum and 90% of PBS) agar media and Littman's agar media. The overnight cultured cells were washed twice with PBS, and 3 mL of cells were spotted on the capsule-inducing media and incubated at 37°C for 2 days. The cells were scraped, resuspended in distilled water, mixed with India ink for visualization, and observed using DIC microscopy. Fifty cells were randomly selected to measure capsule and cell diameter using SPOT Advanced v4.6 software. The capsule thickness was calculated as: total diameter 2 cell body diameter. Statistical significance was calculated using one-way ANOVA analysis with Bonferroni's multiple-comparison test.
Transcriptome analysis using RNA sequencing. Cells cultured overnight were inoculated in fresh YPD broth at the OD 600 of 0.2 and further incubated at 30°C until OD 600 reached 0.8. The cells were pelleted, frozen in liquid nitrogen, and lyophilized overnight. Total RNA was extracted using Easy-BLUE (iNtRON) and purified with a RNeasy minikit (Qiagen). Total RNA was prepared from three independent cultures of each strain. The cDNA library was constructed with the 1 mg of total RNA for each sample using Illumina TruSeq mRNA library kit (Illumina) and sequenced through the Illumina platform. The adapter sequences were trimmed from the sequencing reads using Cutadpat v3.4 with Python v3.7.4. (38). The reference genome sequence of Cryptococcus neoformans H99S and annotation data were downloaded from the NCBI FTP server. The reads were aligned to the C. neoformans H99S genome sequence using Hisat2 v2.2.1 with the Hisat and Bowtie2 algorithm and processed as previously reported (39). Hisat2 was performed with the "-p 30" and "dta -1" option and other parameters set as default. Aligned reads were converted and then sorted using Samtools v1.9 (40) with the "-Sb -@ 8" option for converting and "-@ 20 -m 2000000000" option for sorting with the other parameters set as default. Transcript assembly and abundance estimation were performed using Stringtie v2.1.1 using the "-p 12" and "-B" option to run the Ballgown analysis (41). The assembled transcripts were merged in a single GTF file, and the relative transcript abundances were calculated via fragments per kilobase of exon per million fragments mapped (FPKM). The FPKM and read count matrix were generated using the R package "isoformswitchanalyzerR" and analyzed using DESeq2. Differentially expressed genes (DEG) analysis was performed using DESeq2 v1.24.0 with default sets with the Ballgown. The volcano plot was illustrated using R v4.1.0, with the cutoff: .2-fold changes with P , 0.05. The RNA-seq data were deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE208333), and the differentially expressed genes are listed in Data Set S2.
Flow cytometry analysis. Wild type and KEOPS mutants were cultured to an OD 600 of 0.8, harvested, and washed with PBS. For ethanol fixation, 10 6 cells in 300 mL of PBS were slowly mixed with 700 mL of 100% EtOH to a final concentration of 70% and incubated for 14 to 16 h at 4°C. Fixed cells were pelleted and serially washed with PBS containing 1% and 0.5% BSA. Then the cells were treated with 200 mg/mL RNase (Thermo Scientific, cat. no. EN0531) for 30 min at 37°C. After centrifugation, the cells were stained with propidium iodide staining buffer (100 mg/mL propidium iodide, 100 mM Tris [pH 7.4], 150 mM NaCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% Nonidet P-40) for 2 h at room temperature in the dark. The cells were washed with PBS and filtered through a strainer. Fluorescence was measured with a BD FACS Aria III. We analyzed 30,000 events for each sample.
Primer extension assay. ) cells grown overnight in YPD at 30°C were inoculated into 25 mL fresh YPD medium to an OD 600 of 0.3 and further grown for 3 h. The cell culture was washed thrice with distilled water and collected into a screw-cap tube after centrifugation. All cell pellets were frozen at 280°C until further use. To isolate total RNA, the TRIzol reagent (Invitrogen) was used following the manufacturer's instructions. In brief, the frozen cell pellets were resuspended in 1 mL of TRIzol reagent, and 200 mL of chloroform and acidic glass beads were added. Then the cells were disrupted by shaking and incubated at room temperature for 5 min. Following centrifugation, 500 mL of the aqueous phase was collected into a new tube, and 50 mL of sodium acetate (3 M, pH 5.3) was added. RNA was precipitated with 500 mL of ice-cold isopropanol, washed with ice-cold 70% ethanol, and air-dried. The RNA pellet was dissolved in DEPC-treated water. The concentration and purity of total RNA samples were measured using NanoDrop spectrophotometers (NanoDrop 2000). Following this, 3 mg of total RNA was used in primer extension reactions. The Cn_Ile-AAT-R primer (59-ACGGGATCGAACCGCCGACC -39) and Cn_Val-AAC-R primer (59-CGGGCGGGATCGAACCGCCGACC -39) were labeled at the 59-end with [g-32 P] ATP (6000 Ci/mmol, 10 Ci/L) (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). RNA and the labeled primers were denatured at 65°C for 5 min and then annealed by cooling to 37°C for 90 min. They were then extended at 42°C for 1 h with 5 units (U) of avian myeloblastosis virus reverse transcriptase (AMV RTase) (New England Biolabs). The products were separated on 15% polyacrylamide gel containing 8 M urea. Sequencing ladders were generated using 5 mg of the PCR product amplified from the cDNA of tRNA Ile(AAU) and tRNA Val(AAC) . Images were analyzed in a Bio-Rad phosphorimager using Quantity One software (Bio-Rad Laboratories).
Ergosterol assay. Ergosterol content was measured as previously described (42). Overnight cultured cells were subcultured with fresh YPD broth with OD 600 = 0.2. After OD 600 reached 0.8, the cells were divided into two groups (treated or untreated with 10 mg/mL fluconazole) and further incubated for 24 h. Then the cell pellet was washed with distilled water, frozen in liquid nitrogen, and lyophilized overnight. The dried cell weight was recorded for normalization of ergosterol content. The pellet was added with 5 mL of 25% alcoholic potassium hydroxide (25 g of KOH and 35 mL of sterile distilled water made up to 100 mL with 100% ethanol) and vortexed for 1 min. The sample was transferred to a sterile borosilicate glass screw-cap tube and incubated at 80°C for 1 h. After the sample cooled down at room temperature, 1 mL of sterile water and 3 mL of heptane were added, mixed, and vortexed for 3 min. The 200 mL of the top layer (heptane layer) was harvested and mixed with 800 mL of 100% ethanol. Each 1 mL sample was measured at OD 281.5 and OD 230 . Ergosterol content was calculated as follows: % ergosterol = ([OD281.5/ 290] Â F)/pellet weight 2 ([OD230nm/518] Â F)/pellet weight, where F is the ethanol dilution factor.
Cloning, protein expression, and purification of recombinant Bud32. The full-length BUD32 gene was amplified using PCR from synthesized DNA (Cosmogene tech.) and cloned into the pVFT3S vector (Korean patent 10-0690230), which contains a tobacco etch virus (TEV) protease recognition site (ENLYFQG) between the N-terminal 6ÂHis-thioredoxin (Trx) and multicloning site. The pVFT3S-BUD32 plasmid was inserted into BL21(DE3) via transformation and grown in a high-salt LB medium until OD 600 reached 0.6 to 0.8 at 37°C. The temperature was then lowered to 17°C, and overexpression was induced using 0.3 mM 1thio-b-D-galactopyranoside (IPTG) for 16 h. The 6ÂHis-Trx-Bud32 recombinant protein was purified via metal-affinity chromatography using a Ni-NTA column (GE Healthcare) and digested with TEV protease for 16 h at 4°C. After buffer change through dialysis to remove imidazole, the separated Bud32 and 6ÂHis-Trx were reloaded into the Ni-NTA column, and the flow-through fractions were stored.
Autophosphorylation assay. To verify autophosphorylation of recombinant Bud32 protein, recombinant Bud32 (0.2, 0.5, 1, 2, and 4 mg) was assayed by incubation at 37°C for 30 min in a final volume of 20 mL in the presence of 50 mM Tris-HCl, pH 7.5, 10 mM MnCl 2 , and ATP (50 mM or 100 mM). The reaction was stopped by adding SDS-PAGE loading buffer, and samples were subjected to 12% SDS-PAGE at 50 V for 35 min followed by 100 V for 80 min. To visualize the autophosphorylated proteins, the protein gel was stained with Pro-Q Diamond phosphoprotein gel stain (Invitrogen, P33300) and imaged using Chemi-Doc (Bio-Rad).
In vitro kinase assay. The ATPase/kinase activities of recombinant Bud32 and kinase-dead Bud32 (Bud32 K54A ) proteins were measured using the ADP-Glo kinase assay kit (Promega) according to the instructions provided by the company. The kinase reactions of 4 mg rBud32 or rBud32 K54A were performed with (kinase activity) or without 2 mg of a-casein (ATPase activity), and 100 mM ATP in kinase reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl 2 ) for 30 min at 37°C. Then the kinase reactions were added with 25 mL of ADP-Glo reagent and further incubated for 40 min to stop the kinase reactions and eliminate remaining ATP. Subsequently, 50 mL of kinase detection reagent was added and incubated for 30 min to convert ADP to ATP and introduce luciferase and luciferin to detect ATP. To record luminescence signal, VICTOR X5 multilabel plate reader (PerkinElmer) was used.
Insect-killing assay. The virulence assay using an insect host was performed as previously described (43). Galleria mellonella caterpillars (Vanderhorst Wholesale, Inc., Saint Marys, OH, USA) from final larvae weighing 200 to 300 mg were used within 7 d of arrival for the virulence assay. Each C. neoformans strain was incubated in YPD broth at 30°C overnight, washed thrice with PBS, and adjusted to the final concentration of 10 6 cells/mL in PBS. Four thousand C. neoformans cells were injected into the larvae using repeating dispenser (PB600-1, Hamilton Company, Reno, NV, USA). PBS was injected into larvae as noninfection control. Infected larvae were placed in petri dishes and incubated at 37°C in a humid chamber. The larvae condition was monitored, and their numbers were counted daily. Larvae that turned black and were motionless were considered dead. Pupae during the experiment were censored for statistical analysis.
Murine infectivity assay. Seven-to 8-week-old female C57BL/6 mice purchased from BioLasco Inc. (Taiwan) were used for the murine intranasal infection model (n = 7 for each group) to test the virulence of the wild-type (H99S), bud32D (YSB1968), bud32D::BUD32 (YS B4662), and bud32D::BUD32 K54A (YSB5874) strains. Each strain was grown overnight in YPD broth at 30°C and washed twice with PBS. Afterward, cells were resuspended in PBS and diluted to 10 6 cells/mL. CFU and cell viability were verified by incubating cells on YPD agar at 30°C for 48 h. Mice were anesthetized with isoflurane (Panion & BF Biotech Inc., Taiwan) and infected via intranasal instillation of 50 mL cell suspension (5 Â 10 4 cells). Mice survival was monitored twice daily, and moribund mice (mice that were unable to eat or drink, had a 20% reduction in body weight, or were hunched) were sacrificed. The survival data were plotted using Kaplan-Meier curves and statistically analyzed through log-rank (Mantel-Cox) test using Graph Pad Prism v5.03.
Data availability. We will provide any strain and materials used in this study upon request. RNAseq-based transcriptome profiling data for wild type, and bud32D, bud32D::BUD32 K54A , and kae1D mutants were submitted to Gene Expression Omnibus (accession no. GSE208333).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. DATA SET S1, XLSX file, 0.01 MB. DATA SET S2, XLSX file, 1.8 MB.