Identification of a putative α-galactoside β-(1 → 3)-galactosyltransferase involved in the biosynthesis of galactomannan side chain of glucuronoxylomannogalactan in Cryptococcus neoformans

The cell surface of Cryptococcus neoformans is covered by a thick capsular polysaccharide. The capsule is the most important virulence factor of C. neoformans; however, the complete mechanism of its biosynthesis is unknown. The capsule is composed of glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). As GXM is the most abundant component of the capsule, many studies have focused on GXM biosynthesis. However, although GXMGal has an important role in virulence, studies on its biosynthesis are scarce. Herein, we have identified a GT31 family β-(1 → 3)-galactosyltransferase Ggt2, which is involved in the biosynthesis of the galactomannan side chain of GXMGal. Comparative analysis of GXMGal produced by a ggt2 disruption strain revealed that Ggt2 is a glycosyltransferase that catalyzes the initial reaction in the synthesis of the galactomannan side chain of GXMGal. The ggt2 disruption strain showed a temperature-sensitive phenotype at 37°C, indicating that the galactomannan side chain of GXMGal is important for high-temperature stress tolerance in C. neoformans. Our findings provide insights into complex capsule biosynthesis in C. neoformans.


Introduction
Cryptococcus neoformans, a basidiomycete yeast, is the primary pathogen responsible for cryptococcosis, a globally prevalent disease that affects immunocompromised individuals, specifically those infected with HIV (Maziarz and Perfect, 2016;Altamirano et al., 2020).Upon pulmonary infection, C. neoformans disseminates to the central nervous system and causes severe meningoencephalitis with a high mortality rate.
Herein, we report that a putative glycosyltransferase belonging to the GT31 family in C. neoformans is involved in GXMGal biosynthesis.Using the cap59 disruption strain (a GXM-deficient strain of C. neoformans) as the parental strain, we constructed disruption strains of GT31 family glycosyltransferases (ggt1, ggt2, and ggt3).A nuclear magnetic resonance and methylation gas chromatographymass spectrometry analysis of the structure of GXMGal produced by the cap59 and ggt2 double-disruptant strain revealed that the galactomannan side chain was reduced or almost completely lost.The ggt2 disruption strain exhibited a temperature-sensitive (Ts) phenotype at 37°C.These results indicate that, in C. neoformans, the galactomannan side chain of GXMGal has an important role in hightemperature stress tolerance.

Strains and medium
The C. neoformans strains used in this study are listed in Supplementary Table S1.The C. neoformans var.grubii H99 strain was obtained from the Fungal Genetics Stock Center (Kansas City, USA).

Construction of the cap59 disruption strain
CAP59 (CNAG_00721) was disrupted in C. neoformans H99 by inserting NEO using the CRISPR/Cas9 system (Huang et al., 2022).A gene replacement cassette encompassing a 50-bp homology arm at the 5′ end and a 50-bp homology arm at the 3′ end of CAP59 was amplified by recombinant PCR using pNEO_6xHA (So et al., 2017) as a template and the cap59-del-F-cap59-del-R primer pair (Supplementary Table S2).The Cas9 expression cassette was amplified by PCR using pBHM2403 as a template and the M13-F-M13-R primer pair.The sgRNA expression cassette was amplified in two PCR steps.An sgRNA scaffold containing a 20-bp target sequence and U6 promoter and an sgRNA scaffold containing a 20-bp target sequence and U6 terminator was amplified by PCR using pBHM2329 as a template and the M13-F-cap59-gRNA-R1 and cap59-gRNA-F2-M13-R primer pairs.The PCR fragments were combined by fusion PCR using the U6-F-U6-R primer pair.All PCR fragments were introduced into C. neoformans by electroporation using Gene Pulser II (Bio-Rad, Hercules, CA), yielding cap59Δ strain.Transformants were selected using YPD agar plates supplemented with 200 μg/ mL G418.The introduction of NEO into each locus was confirmed by PCR using the cap59-comf-F-cap59-comf-R primer pair (Supplementary Figure S1).
2.4 Construction of the cap59, ggt2, and ggt3 triple disruption strain GGT3 was disrupted in C. neoformans cap59Δggt2Δ strain by inserting NAT.A gene replacement cassette encompassing a 50-bp homology arm at the 5′ end and a 50-bp homology arm at the 3′ end of GGT3 was amplified by recombinant PCR using pNAT_mCherry (So et al., 2017) as a template and the ggt3-del-F-ggt3-del-R primer pair (Supplementary Table S2).Transformants were selected on YPD agar plates supplemented with 100 μg/mL nourseothricin sulfate.The introduction of NAT into each locus was confirmed by PCR using the ggt3-comf-F-ggt3-comf-R primer pair (Supplementary Figure S2).

Complementation of the ggt2 disruption strain with wild-type GGT2
For complementation, analysis of GGT2 using a gene replacement cassette, encompassing a homology arm at the 5′ end of GGT2, wildtype GGT2 containing 3′-UTR, hygromycin B resistance gene (hph), and a homology arm at the 3′ end of GGT2, was constructed by recombinant PCR using H99 genomic DNA; pNAT_mCherry as a template; and the ggt2-comp-1-ggt2-comp-2, ggt2-comp-3-ggt2comp-4, and ggt2-comp-5-ggt2-comp-6 primer pairs.The resultant DNA fragment was amplified with the ggt2-comp-1-ggt2-comp-6 primer pair.An sgRNA scaffold containing a 20-bp target sequence and U6 promoter and an sgRNA scaffold containing a 20-bp target sequence and U6 terminator were amplified by PCR using pBHM2329 as a template and the M13-F-HYG-gRNA-R1 and HYG-gRNA-F2-M13-R primer pairs.All PCR fragments were introduced into C. neoformans ggt2Δ and cap59Δggt2Δ by electroporation.Transformants were selected on YPD agar plates supplemented with 100 μg/mL nourseothricin sulfate.The introduction of NAT into each locus was confirmed by PCR using the ggt3-comp-comf-F-ggt3comp-comf-R primer pair (Supplementary Figure S1).

Measurement of capsule size
C. neoformans strains were cultured in 3 mL of YPD liquid medium at 30°C for 24 h.The cells were then collected by centrifugation and washed three times with sterile phosphate-buffered saline (PBS), suspended in 2 mL of 10% Sabouraud liquid medium, and incubated at 30°C for 24 h to induce capsule production.The culture medium was diluted with PBS mixed at a ratio of 1:1 with India ink (Syogeikuretake Shikon BB1-18; Kuretake Co., Ltd.Nara, Japan) and incubated for 15 min.Images of stained cells were acquired using a microscope equipped with a digital camera.The diameters of the cells and capsules were measured immediately (50 cells), and the average diameter was calculated.

Preparation of the GXMGal fraction
Purification of GXMGal was performed as described (Rocha et al., 2015).Briefly, cap59Δ strains were cultivated in 1 L of 10% Sabouraud medium at 30°C with shaking (160 rpm) for 5 days.The culture supernatant was collected by centrifugation, mixed with an equal volume of phenol:chloroform, and centrifuged.The collected supernatant was dialyzed overnight at 4°C using a Visking Tube (Nihon Medical Science, Inc. Japan).The polysaccharides were powdered by lyophilization.GXMGal powder was dissolved in 3% cetyltrimethylammonium bromide solution in 1% borate at pH 9.5.The GM fraction was collected as precipitate, washed with 75% ethanol, dialyzed with water, and lyophilized.

Methylation GC-MS and nuclear magnetic resonance spectroscopy
Glycosidic linkages were analyzed as previously described (Klutts and Doering, 2008;Katafuchi et al., 2017).Briefly, GXMGal was separately dissolved in dimethyl sulfoxide, followed by NaOH addition.After stirring for 3 h, methyl iodide was added, and the suspension was stirred for 24 h.The methylated products were extracted in chloroform and washed using dH 2 O.Then, the methylated samples were hydrolyzed using 2 M trifluoroacetic acid, reduced, and acetylated.The partially methylated alditol acetates were analyzed by GC-MS using a capillary column (30 m × 0.25 mm; DB-5, Agilent, CA) with helium as the carrier gas at a gradient temperature program of 210°C-260°C at 5°C/min.The GC-MS analyses were performed using a JMS-K9 mass spectrometer (JEOL, Tokyo, Japan).NMR experiments were performed as previously described (Klutts and Doering, 2008;Katafuchi et al., 2017).The NMR spectra were recorded using a JNM-LA600 spectrometer (JEOL) at 45°C.Proton and carbon chemical shifts were referenced relative to internal acetone at δ 2.225 and 31.07 ppm, respectively.

Identification of candidate galactosyltransferases involved in GXMGal biosynthesis in C. neoformans
First, we searched the C. neoformans H99 genome for candidate genes encoding enzymes that transfer the β-galactosyl residue to the hydroxyl group at position 3 of the α-galactosyl residue in GXMGal.S. pombe Pvg3 is a glycosyltransferase that exhibits similar enzymatic activity.Therefore, we selected candidate genes by PSI-BLAST search using Pvg3 as a query.Two homologous proteins were selected (CNAG_01050 and CNAG_01385).CNAG_01385 is Ggt1--an α-mannoside β-(1 → 6)-galactosyltransferase involved in GIPC biosynthesis in C. neoformans.Then, a BLASTp search was performed using CNAG_01050 as a query to select candidate genes.CNAG_06918 was selected in addition to CNAG_01385.Therefore, we named CNAG_01050 and CNAG_06918 as Ggt2 and Ggt3, respectively, the two α-galactoside β-(1 → 3)-galactosyltransferase candidates.Ggt2 and Ggt3 are members of the GT31 family.Ggt2 has an amino acid sequence homology of 23% (in the 434-645 amino acid region of Ggt1) and 33% (in the 122-320 amino acid region of Ggt3) with Ggt1 and Ggt3, respectively (Figure 1).Ggt1 does not have a transmembrane domain, but analysis using DeepLoc 2.0 (Thumuluri et al., 2022) predicted a transmembrane region at 338-360 aa.Ggt2 and Ggt3 were predicted to be Golgi-localized type II membrane proteins with one transmembrane region at N-terminal 35-57 and 21-43 aa, respectively, according to DeepLoc 2.0 (Figure 1).

Phenotypic analysis of GGT mutants
To clarify the physiological role of GGT in C. neoformans cells, ggt1, ggt2, and ggt3 single-disruptant strains were constructed using H99 as the parental strain.As uge1Δ and ugt1Δ cannot supply UDP-Gal to the Golgi, they are deficient in GXMGal and GIPC biosynthesis and exhibit a Ts phenotype at 37°C (Moyrand et al., 2008;Li et al., 2017).ggt1Δ lacks GIPC and exhibits a Ts phenotype at 37°C.Therefore, we observed growth of GGT disruptants at 37°C (Figure 3).At 30°C, ggt1Δ showed slightly delayed growth compared with the wild-type; moreover, ggt2Δ and ggt3Δ showed similar growth compared with the H99 strain.By contrast, at 37°C, ggt1Δ and ggt2Δ showed dramatically delayed growth, indicating a Ts phenotype.The growth of ggt3Δ and H99 was similar.These results indicate that the glycan structure synthesized by Ggt2 is important for high-temperature stress tolerance in C. neoformans.

Drug resistance of ggt2 disruptant strain
We examined the growth of ggt2Δ on media containing various drugs.ugt1 disruptant strains show sensitivity to NaCl, Congo red, H 2 O 2 , and sodium dodecyl sulfate (SDS) (Li et al., 2017).Therefore, we tested the sensitivity of ggt2Δ to these drugs (Figure 4).ggt2Δ was not significantly sensitive to any of the drugs.Conversely, the Ts phenotype of ggt2Δ was completely rescued by complementation with wild-type GGT2 and slightly rescued by 1 M sorbitol, which exerts high osmotic pressure.These results indicate that the galactomannan side chain of GXMGal biosynthesized by Ggt2 is important for high-temperature stress tolerance in C. neoformans.

Capsule productivity of ggt2 disruptant strain
To further analyze the ggt2Δ phenotype, the capsule structure was stained with India ink for microscopic observation (Supplementary Figure S3).Quantification of cell and capsule sizes revealed no significant differences between ggt2Δ and the wild-type strain.These findings indicated that Ggt2 absence did not affect GXM production.Frontiers in Microbiology frontiersin.org

Role of Ggt2 in GXMGal biosynthesis
To examine the role of Ggt2 in GXMGal biosynthesis, we complemented wild-type GGT2 in cap59Δggt2Δ and analyzed the structure of GXMGal.The structure of GXMGal produced by each strain was analyzed by 1 H-NMR (Figure 5).Sharp doublet peaks at 4.98 ppm of the α-galactan backbone seen in cap59Δggt2Δ disappeared due to GGT2 complementation, and the Man-derived chemical shift seen in wild-type GXMGal reappeared.

Phylogenetic analysis of Pvg3, Ggt1, Ggt2, and Ggt3 family proteins belonging to the GT31 family
Sequences of Pvg3, Ggt1, Ggt2, and Ggt3 family proteins were used to construct an evolutionary phylogenetic tree (Figure 7).The data set for analysis was obtained from FungiDB using the amino acid sequences of S. pombe Pvg3 and C. neoformans Ggt1, Ggt2, and Ggt3 as search queries. 1 The protein sequences were clearly divided into the 1 https://fungidb.orgPhenotype of ggt1, ggt2, and ggt3 single disruptants.Colony morphology of H99, ggt1Δ, ggt2Δ, and ggt3Δ cultured on YPD agar at 30°C and 37°C for 3 days, respectively.The agar medium was inoculated with 10-fold serial dilutions of cells adjusted to 10 6 cells.
This study aimed to identify glycosyltransferases involved in the biosynthesis of C. neoformans capsules.We identified a putative β-(1 → 3)-Gal transferase belonging to the GT31 family that plays a role in GXMGal biosynthesis (Figure 8).Ggt1 is conserved in a wide range of species in the phylum Basidiomycota, whereas Ggt2 is only conserved in certain basidiomycete yeasts, such as Pucciniomycetes and Tremellomycetes (Figure 7).Multiple alignments of Ggt2 homologs revealed that amino acids in the GT-A domain are highly conserved (Supplementary Figure S4).Therefore, Ggt2 homologs may be responsible for synthesizing important glycan structures, including GXMGal.The enzyme involved in capsule biosynthesis has attracted attention as a novel antifungal drug target due to its contribution to virulence (Almeida et al., 2015).Many putative glycosyltransferases, involved in GXM biosynthesis, have been identified as CAP genes, because they can be easily screened based on phenotypes, such as India ink-negative staining (Fromtling et al., 1982).However, identification of glycosyltransferases involved in capsule biosynthesis is challenging, and only a few glycosyltransferases have been identified: α-(1 → 3)-Man transferase Cmt1 (Doering, 1999;Sommer et al., 2003) is involved in GXM biosynthesis and β-(1 → 2)-Xyl transferases Cxt1 and Cxt2 are involved in GXMGal biosynthesis (Klutts et al., 2007;Klutts and Doering, 2008;Reilly et al., 2009;Wang et al., 2018).We believe that our discovery of Ggt2 will contribute to studies on capsule biosynthesis in C. neoformans.Drug sensitivity of the ggt2 disruptant.Colony morphology of H99, ggt2Δ, and ggt2Δ + GGT2 on YPD agar supplemented with or without 1 M sorbitol, 1 M NaCl, 1 mg/mL calcofluor white (CFW), 1 mg/mL Congo red (CR), 5 mM H 2 O 2 , and 0.005% sodium dodecyl sulfate (SDS) at 30°C and 37°C for 3 days.The agar medium was inoculated with 10-fold serial dilutions of cells adjusted to 10 6 cells.Methylation GC-MS NMR analyses revealed the detailed role of Ggt2 in GXMGal biosynthesis (Table 1; Figure 6).The loss or severe reduction of the galactomannan side chain of GXMGal in the ggt2 disruptant strain indicates that Ggt2 is the only α-Gal β-(1 → 3)-Gal transferase involved in GXMGal biosynthesis (Figure 2) because Ggt1 and Ggt2 accept α-mannoside and α-galactoside as receptor substrates, respectively.This is a logical result because the structure of the sugar chains involved in their biosynthesis suggests that Ggt1 and Ggt2 are likely to use α-mannoside and α-galactoside as their acceptor substrates, respectively.The GXMGal structure between the ggt3Δ and wild-type strains was not notably different and did not exhibit a Ts phenotype, suggesting that Ggt3 is not involved in GXMGal or GIPC biosynthesis (Figures 3, 4).The function of Ggt3 must be analyzed in detail.Interestingly, methylation GC-MS analysis detected methylesterified sugars that may have originated from glycans other than GXM and GXMGal.These indicate the presence of unknown glycan structures, such as N-or O-glycans or glycolipids.Considering how GXMGal was first discovered in the culture supernatant of a mutant strain lacking GXM (Cherniak et al., 1982), C. neoformans may possess unknown glycan structures.Thus, the detailed structures of these glycans should be clarified in the future.
Phenotypic analysis of gene disruptant strains revealed the physiological functions of Ggt2.ggt2Δ, like ggt1Δ, exhibited a Ts phenotype at 37°C, indicating that the galactomannan side chain of GXMGal, like GIPC, is important for high-temperature stress tolerance in C. neoformans.Notably, GXMGal is important for hightemperature stress tolerance in C. neoformans, although it is less abundant than GXM.In C. neoformans, Saccharomyces cerevisiae, and Aspergillus fumigatus, O-glycan deficiency leads to reduced hightemperature stress tolerance and disruption of cell wall integrity (Wagener et al., 2008;Kadooka et al., 2022;Thak et al., 2022).However, ggt2Δ was insensitive to Congo red or calcofluor white, which are inhibitors of cell wall synthesis, suggesting that loss of galactomannan side chains in GXMGal does not affect cell wall integrity in C. neoformans.Additionally, ugt1Δ is sensitive to Congo red, NaCl, and SDS (Li et al., 2017), but ggt2Δ is not, suggesting that  phenotype of ugt1Δ is not due to the loss of galactomannan side chains of GXMGal but rather due to a loss of GIPC.Consistently, GXM-deficient mutants were sensitive to NaCl and SDS (Li et al., 2018a,b).The phenotypic differences in polysaccharide-deficient mutants are interesting and should be analyzed further.
We used a bacterial heterologous expression system to generate recombinant Ggt2 and measured its β-Gal transfer activity to 4-methylumbelliferylated α-Gal.However, we could not detect glycosyltransfer activity (data not shown).This may be characteristic of the substrate specificity of Ggt2.The galactomannan side chain of GXMGal is added in succession, probably because Ggt2 recognizes di-or trisaccharide α-galactooligosaccharides as substrates and can only transfer β-Gal to a certain location.Another hypothesis is that Ggt2 does not exhibit glycosyltransferase activity by itself.For example, S. pombe Pvg3, which belongs to the GT31 family, is not active alone but exhibits glycosyltransferase activity by forming a complex with several proteins (Fukunaga et al., 2023).The enzymatic features of Ggt2 may be essential for the formation of the unique GXMGal structure and should be studied in more detail.
In conclusion, we have partially identified the mechanism of GXMGal biosynthesis in C. neoformans.Our findings will contribute substantially to understanding the structure and biosynthesis of the fungal cell wall and developing anticryptococcal agents.Phylogenetic analysis of Ggt1, Ggt2, and Ggt3 homologs in basidiomycetes and Pvg3 homologs in basidiomycetes and fission yeasts.Protein sequences were downloaded from FungiDB.The phylogenetic tree was drawn using iTOL.Alignment and phylogenetic tree inference were performed using MAFFT and RAxML,respectively,included in ETE v3.note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

FIGURE 1
FIGURE 1 Schematic of the Ggt1, Ggt2, and Ggt3 proteins.The vertical black bars indicate transmembrane (TM) domains of Ggt1 (338-360 aa), Ggt2 (35-57 aa), and Ggt3 (21-43 aa).The gray bars indicate GT-A fold domains of Ggt1, Ggt2, and Ggt3.The dark gray bar indicates an unknown domain of Ggt1.The identity of the amino acid sequences of Ggt1 and Ggt2 and Ggt2 and Ggt3 in the GT-A fold domain are indicated.

TABLE 1
Methylation analysis of GXMGal from the ggt2 disruptant.
b n.d.means none detected.