Filamentous Fungi

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan, and glycosyl inositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple galactofuranose monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in galactofuranose (Galf) antigen biosynthesis. Disruption of gfsA reduced binding of '-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro galactofuranose antigen synthase assay revealed that GfsA has '1,5- or '1,6- galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-D-galactofuranose as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. 'gfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature, and limited formation of conidia. Several gfsA orthologs were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal '-galactofuranosyltransferase, which was shown to be involved in galactofuranose antigen biosynthesis of O-glycans in the Golgi.

Introduction
Polysaccharides and glycoconjugates are present in the plasma membrane, cell wall, and extracellular environment, and play important roles in many biological events. Filamentous fungal cell walls contain many polysaccharides, including '1,3-, '1,6-, and '1,3-linked glucans, chitin, galactosaminogalactan, and galactomannan, as well as O- and N-glycans linked to proteins (Bernard, 2001; de Groot, 2009; Gastebois, 2009; Latg, 2009; Latg, 2010; Fontaine, 2011; Jin, 2012). The components and polymeric organization of the Aspergillus cell wall have been determined, and several glycosyltransferase genes involved in the synthesis of these components have been identified and characterized (Kelly, 1996; Motoyama, 1997; Horiuchi, 1999; Shaw, 2002; de Groot, 2009; Yoshimi, 2013).

Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in a number of filamentous fungi, including various Aspergillus species, bacteria, trypanosomatids, and nematodes, but is absent in yeast, mammals, and plants (Latg, 2009; Tefsen, 2012). Galf residues are immunogenic in mammals (Reiss, 1979; Yuen, 2001; Morelle, 2005) and are speculated to be involved in pathogenicity in humans. Therefore, the inhibition and detection of the biosynthesis of Galf might be exploited for chemotherapy and as a diagnostic tool for Aspergillus infection (Pedersen, 2003; Latg, 2009; Tefsen, 2012).

Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans, which modify many cell wall proteins and extracellular enzymes (Wallis, 1999; Wallis, 2001; Goto, 2007; Tefsen, 2012). A single 1,2-linked Galf residue is present at the non-reducing terminus of N-glycans (Takayanagi, 1994; Wallis, 2001; Morelle, 2005; Schmalhorst, 2008), and terminally '1,5-linked Galf residues are present in O-mannose glycans (Leitao, 2003) (Fig. 1). O-mannose glycan modification of proteins is widely distributed in bacteria and eukaryotic cells, from yeast to mammals, and is required for the formation of filamentous fungal cell walls (Shaw, 2002; Oka, 2004; Oka, 2005; Zhou, 2007; Mouyna, 2010; Goto, 2009; Kriangkripipat, 2009; Lommel, 2009). The initial reaction in the transfer of mannose to serine or threonine residues is catalyzed in the endoplasmic reticulum by protein-O-D-mannosyltransferase (Pmt), which requires dolichol phosphate-mannose as an immediate sugar donor (Strahl, 1999). Although O-glycans are elongated by glycosyltransferases in the Golgi apparatus, the responsible enzymes, such as galactofuranosyltransferases, remain to be identified in filamentous fungi.

Galf is also found in fungal-type galactomannan of Aspergillus (Fig. 1). Fungal-type galactomannan of Aspergillus is composed of a linear mannan core with an '1,2-linked mannotetraose repeating unit attached via an '1,6-linkage and '1,5-Galf oligomers (galactofuran side chain) of up to five residues that are attached to the mannan backbone via '1,6- or '1,3-linkages (Latg, 1994; Fig. 1). Galactomannan is anchored to the plasma membrane by glycosylphosphatidylinositol and is covalently bound to the non-reducing end of a short '1,3-glucan chain in the cell wall (Fontaine, 2000; Costachel, 2005). However, galactomannan has not been identified in the glycosylphosphatidylinositol anchors of Aspergillus spp. (Tefsen, 2012).

Although little is known about the enzymes involved in Galf initiation, extension, and termination, several enzymes involved in the synthesis of Galf-containing sugar chains have been studied in fungi. For example, UDP-glucose 4-epimerase (UgeA) is responsible for the conversion of UDP-glucose to UDP-galactopyranose (Galp) (El-Ganiny, 2010), which is then converted to UDP-Galf by UDP-Galp mutase (UgmA/GlfA) in the cytosol (Bakker, 2005; Damveld, 2008; El-Ganiny, 2008; Schmalhorst, 2008). The synthesized UDP-Galf is then transported into the Golgi lumen by the Golgi-localized UDP-Galf transporter (UgtA/GlfB) (Engel, 2009; Afroz, 2011), which is unique to Aspergillus spp. and is required for Galf deposition in the cell wall. UDP-Galf plays an important role in the Galf biosynthetic pathway, where it acts as a sugar donor for galactofuranosyltransferases (Engel, 2009; Afroz, 2011). An Aspergillus fumigatus glfA deletion mutant strain was shown to lack Galf residues and displayed attenuated virulence in a mouse model of invasive aspergillosis (Schmalhorst, 2008). In contrast, Lamarre (2009) reported that glfA deletion in a different A. fumigatus strain results in increased adhesion to host cells due to a lack of galactofuran, and no attenuation of virulence. Heesemann (2011) reported that the monoclonal antibody IgM L10-1, which specifically reacts with Galf-containing glycostructures, fails to inhibit hyphal growth of A. fumigatus and is not able to protect infected mice. Furthermore, Aspergillus nidulans Galf-deficient mutants exhibit compact colony growth, and abnormal conidiation and hyphal morphology (El-Ganiny, 2008). Taken together, these results suggest that Galf residues play important roles in cell wall integrity and pathogenicity in Aspergillus spp. To elucidate the individual role of Galf-containing polysaccharides and glycoconjugates in cell wall biosynthesis, it is necessary to identify and characterize the galactofuranosyltransferases that catalyze the addition of Galf residues from UDP-Galf.

Several bacterial genes reportedly encode galactofuranosyltransferases. For example, the product of the glfT gene is required for mycobacterial galactan polymerization and has both '1,5- and '1,6-Galf transferase activities (Kremer, 2001). In addition, WbbI in Escherichia coli performs '1,6-coupling of Galf to '-glucose (Wing, 2006) and WbbO in Klebsiella pneumoniae couples Galf to Galp by a '1,3-linkage (Guan, 2001). To date, LPG1 of Leishmania sp. is the only Galf transferase described in eukaryotes and the encoding gene was isolated by functional complementation of the Leishmania donovani R2D2 mutant (Ryan, 1993). Evidence suggests that LPG1 adds '1,3-Galf residue onto mannose (Huang, 1993). Although many laboratories have likely searched for galactofuranosyltransferases involved in the synthesis of Galf-containing polysaccharides and glycoconjugates, to our knowledge, the identification of such an enzyme has not been reported.

In the present report, we attempted to identify galactofuranosyltransferase genes involved in the synthesis of Galf antigen in A. nidulans and A. fumigatus using reverse-genetic and biochemical approaches. We speculated that galactofuranosyltransferase candidate genes might be among the set of A. nidulans genes encoding unidentified and uncharacterized putative glycosyltransferases that are not orthologs of genes identified in other organisms. By constructing and screening a gene-disruptant library of candidate genes involved in the biosynthesis of Galf antigen, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen of O-glycan synthesis. Sucrose density gradient centrifugation analysis revealed that Galf is localized to Golgi apparatus, similar to the UDP-galactofuranose transporter UgtA/GlfB (Engel, 2009; Afroz, 2011). Based on these results, we updated the biosynthetic pathway for Galf antigen in Aspergillus to include the galactofuranosyltransferase GfsA.

Results
Selection of candidate genes involved in the synthesis of galactofuranose antigens - A BLAST search (Altschul, 1997) of the Aspergillus genome did not identify any orthologs of Leishmania or bacterial galactofuranosyltransferase genes. Thus, we used a reverse-genetic approach that did not rely on primary sequence similarity and instead was based on the fact that galactofuranose residues are found in some filamentous fungi, protozoa, and nematodes (Beverley, 2005), but not in yeasts, mammals, or plants (Tefsen, 2012). A search of the CAZy database (carbohydrate-active enzymes database; http://www.cazy.org/) revealed that approximately 90 genes in the A. nidulans genome encode putative glycosyltransferases (Campbell, 1997). We selected candidate genes that might be involved in the synthesis of Galf antigen using the following approach. First, we excluded glycosyltransferase family 1 (GT1) genes, which are thought to be involved in the synthesis of glycosides. Second, we excluded orthologs of genes identified in other organisms, including Saccharomyces cerevisiae. Finally, we selected genes common to filamentous fungi that contain Galf antigen in their cell walls. Based on these criteria, 11 candidate genes encoding putative galactofuranosyltransferases in A. nidulans were selected (Table S1).

Screening of genes involved in the synthesis of galactofuranose antigen - To analyze the function of the 11 putative glycosyltransferase-encoding genes, each gene was disrupted in A. nidulans AKU89 by gene replacement with argB (Table S1 and Table S2). Chromosomal targeting of the 11 genes was confirmed by PCR using the primers ANxxxx-1 and argB-R, and argB-F and ANxxxx-4 (Yu, 2004; Table S4, Supplemental Fig. 1A and 1B), and at least two independent isogenic disruptant strains were isolated for each candidate gene. The colony phenotypes of all disruptant strains were observed after 3 days of growth at 30C on minimal medium. Notably, strain 'AN8677 formed small white colonies, a phenotype similar to that of strain 'ugmA, which lacks Galf (Fig. 2A; El-Ganiny, 2008; Alam, 2012).
We next tested the affinity of EB-A2, a monoclonal antibody that binds Galf residues with high specificity, towards galactomannoproteins extracted from the disruptant strains (Fig. 2B). Clinical screening with EB-A2 is considered to be a highly sensitive and specific assay for early detection of invasive aspergillosis (Klont, 2004), and the specificity of EB-A2 for Galf antigen is well characterized (Stynen, 1992; Yuen, 2001; Leitao, 2003; Morelle, 2005). The main epitope of EB-A2 is reportedly a tetrasaccharide of '1,5-linked Galf (galactofuran side chain) (Stynen, 1992); however, EB-A2 also reacts with a single terminal non-reducing Galf residue in N-glycans and terminal '1,5-linked Galf residues in O-glycans (Yuen, 2001; Leitao, 2003; Morelle, 2005). Based on these characterizations, EB-A2 is considered to represent a selective anti-'-Galf antibody.
Previous work has shown that EB-A2 bound to cell wall proteins of Aspergillus spp. is detectable as a smear that migrates between 40 to >200 kDa, whereas binding is absent in Galf-defective mutants (Stynen, 1992; Schmalhorst, 2008; Engel, 2009; Afroz, 2011). Here, immunoblot analysis was used to examine the binding of EB-A2 to galactomannoproteins extracted from the 11 A. nidulans disruptants (Fig. 2B). The analysis revealed a marked reduction in the EB-A2 signal for strain 'AN8677 compared to wild type (wt), suggesting that AN8677 is involved in the biosynthesis of Galf antigen (Fig. 2B). Detection of EB-A2-positive signals in the wt and other disruptant strains demonstrated that Galf antigen was present (Fig. 2B).
We next measured the levels of D-galactopyranose in galactomannoproteins extracted from the wt and 'AN8677 strains. It is generally known that furanoses, included in polymers, are detected as pyranoses after hydrolysis. The D-galactopyranose content of strain 'AN8677 was only 70% of that observed for wt (Fig. 2C), providing further evidence that the levels of Galf antigen are reduced in strain 'AN8677. Taken together, these results suggested that AN8677 is involved in the biosynthesis of Galf antigen and was therefore named gfsA (galactofuranose antigen synthase).
Southern blotting was used to examine the structure of the gfsA locus in strain 'AN8677 ('gfsA) (Fig. 3). The analysis revealed that site-specific recombination with the argB cassette occurred at the chromosomal gfsA locus (Fig. 3A and 3B). Next, we introduced the entire gfsA gene from A. nidulans into strain 'gfsA, yielding strain 'gfsA+pPTR-II-gfsA. As expected, introduction of A. nidulans gfsA into 'gfsA rescued the abnormal colony phenotype and conidia formation at 30C (Fig. 3C). In addition, Galf antigen in galactomannoproteins from strain 'gfsA+pPTR-II-gfsA was detected by immunoblotting with EB-A2 at signal intensities that were comparable to wt (Fig. 3D). These results are consistent with the speculation that gfsA is responsible for the defect in strain 'gfsA.
To investigate the origin of the signal associated with the binding of EB-A2, we performed immunoblot analysis with proteinase K-treated galactomannoproteins extracted from wt (Supplemental Fig. 2). We found that EB-A2 signal intensity decreased after proteinase K treatment, indicating that the signal originated from Galf antigen attached to galactomannoproteins (Supplemental Fig. 2).
To determine if Galf residues are attached to N-glycans and O-glycans in galactomannoproteins extracted from the wt strain, we first released N-glycans and O-glycans from galactomannoproteins by PNGase F treatment and '-elimination, respectively (Fig. 4A). Treatment of galactomannoproteins with PNGase F did not affect EB-A2 signal intensity, suggesting that the signal is not related to the Galf residues attached to N-glycans (Fig. 4A, lanes 1 and 2). By contrast, '-elimination treatment of galactomannoproteins resulted in a decrease in signal intensity (Fig. 4A, lanes 1, 3, and 4). Next, we tested the reactivity of EB-A2 to proteins extracted from A. nidulans strains disrupted for pmtA and pmtC, which encode enzymes that initiate the synthesis of O-mannose-type glycans (Shaw, 2002; Oka, 2004; Goto, 2009; Kriangkripipat, 2009). These disruptants have reduced levels of protein O-mannosylation (Oka, 2004; Goto, 2009; Kriangkripipat, 2009). We found that the signal intensity of both 'pmtA and 'pmtC strains was lower as compared to wt, indicating that the signal originated from Galf residues in O-glycans (Fig. 4B). In addition, we confirmed that Galf residues in O-mannosylated proteins were reduced in the 'gfsA strain as compared with wt (Fig. 4B).
We previously reported that A. nidulans WscA, a putative stress sensor protein, is N- and O-glycosylated (Goto, 2009; Fugatami, 2011). We therefore constructed wt, 'gfsA, and 'ugmA strains expressing a WscA-HA fusion protein, which was then purified from each strain subjected to immunoblotting analysis with anti-HA antibody and EB-A2 after PNGase F treatment (Fig. 4C). The EB-A2 signal intensity of WscA-HA from 'gfsA was less than that from wt (Fig. 4C, lanes 4 and 5). Densitometric quantification of the immunoblot bands was performed using Quantity One Ver. 4.4.1 (Bio-Rad). The signal intensity ratios for EB-A2 and anti-HA were then calculated and the ratio for wt was normalized to 1.0. The ratio of intensities of EB-A2 to anti-HA from 'gfsA was less than half of that from wt (Fig. 4D). These data indicate that the gfsA gene is involved in the synthesis of the Galf antigen in O-glycans.

Features of GfsA - The gfsA gene encodes a 532-amino acid protein with a putative molecular mass of 60.9 kDa (Supplemental Fig. 3). gfsA cDNA was amplified by PCR using a 24-h developmental cDNA library (obtained from the FGSC) as a template. Comparison of the cDNA and genome sequences revealed that no introns were present in the gfsA gene. Analysis of secondary structure using SOSUI (Hirokawa, 1998) revealed that GfsA has a putative signal sequence (amino acids 1-17) and transmembrane domain (amino acids 18'40) at the N-terminus, suggesting that GfsA is a type II membrane protein. GfsA also has a metal-binding DXD motif (amino acids 256'258), which is conserved among GfsAs from other organisms, and two potential N-glycosylation sites (amino acids 93'95 and 414'416) (Supplemental Fig. 3). Two putative paralogs, AN5663 and AN2015, were identified that have 27.5% and 26.0% sequence identity, respectively, with GfsA. However, disruption of these genes in A. nidulans did not result in any alteration of the colony phenotype under our experimental conditions (Fig. 2A).
BLAST analysis indicated that GfsA proteins are widely distributed in the subphylum Pezizomycotina (Table S3). However, no orthologs of GfsA were found in the genomes of Caenorhabditis elegans, Trypanosoma cruzi, or bacteria, including E. coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis. GfsA has 87% sequence identity with A. flavus NRRL 3357 (AFL2G_12474), 84% with A. niger CBS 513.88 (An12g08720), 84% with A. fumigatus A1163 (AFUB_096220), 84% with Af293 (Afu6g02120), 83% with A. oryzae RIB40 (AO090120000096), 76% with Penicillium chrysogenum Wisconsin 54-1255 (Pc22g24320), 62% with Ajellomyces capsulatus NAm1 (HCAG_03193), 57% with Coccidioides immitis RS (CIMG_05833), 56% with Paracoccidioides brasiliensis Pb03 (PABG_06492), 55% with Trichophyton rubrum CBS118892 (TERG_00129), 32% with Neurospora crassa OR74A (NCU02213), and 32% with Fusarium graminearum PH-1 (FGSC_05770).

Structural and functional analyses of GfsA - To enable a better understanding of GfsA, we constructed a strain expressing 3xFLAG-tagged GfsA by chromosomal tagging (Fig. 5A). The phenotype of the tagged strain was identical to wt, indicating that 3xFLAG-tagged GfsA has similar functional to that of wt GfsA. To confirm expression of 3xFLAG-tagged GfsA, a solubilized protein was prepared from the tagged strain and analyzed with anti-FLAG antibody (Fig. 5B). The strain expressing 3xFLAG-tagged GfsA produced a protein of approximately 67 kDa (Fig. 5B, lane 1). After endoglycosidase H (Endo Hf) treatment, 3xFLAG-tagged GfsA showed faster mobility during SDS-PAGE, with an apparent molecular weight of approximately 61 kDa (Fig. 5B, lane 2). These results show that GfsA is N-glycosylated.
To determine the enzymatic function of GfsA, we assayed Galf antigen synthase activity using an in-vitro assay that was developed for measuring galactofuranosyltransferase activity using EB-A2 antibody. To exclude the influence of intracellular Galf antigen, the assay was conducted in the 'ugmA background ('ugmA and GfsA-3xFLAG ('ugmA) strains). GfsA proteins were first immunoprecipitated from strains GfsA-3xFLAG ('ugmA) and 'ugmA (negative control) using anti-FLAG beads and then subjected to SDS-PAGE and silver staining (Fig. 5C, left panel). Purified GfsA was detected as two distinct protein bands of approximately 67 kDa (Fig. 5C, lane 1). The upper 67-kDa band is intact GfsA-3xFLAG and the lower band is a degradation product that retained reactivity to HA antibody (Fig. 5C, lanes 1 and 3). Although a few weakly stained bands of lower molecular weight can be observed in the gel, these bands were also present in the control sample. These results indicate that GfsA-3xFLAG was highly purified.
Galactofuranosyltransferase activity of the purified protein was assayed using 0.72 mM UDP-Galf as a sugar donor, 0.5 mM Mn2+ as a co-factor, and 2.5 g of galactomannoproteins from strain 'gfsA (GMP'gfsA). A strong signal of EB-A2 binding was detected in immunoblots when purified GfsA-3xFLAG was used (Fig. 5D, lane 1), whereas no detectable galactofuranosyltransferase activity was observed in the control fraction (Fig. 5D, lane 2). These results indicated that the purified GfsA fraction was not contaminated with any other galactofuranosyltransferase and that GfsA has galactofuranosyltransferase activity. To confirm these results, we also assayed the galactofuranosyltransferase activity of solubilized proteins extracted from 'ugmA and 'ugmA'gfsA (Supplemental Fig. 4). A signal intensity of EB-A2 staining on immunoblots was only detected for solubilized proteins extracted from 'ugmA (Supplemental Fig. 4, lane 1). In contrast, the solubilized protein fraction obtained from 'ugmA'gfsA did not have any galactofuranosyltransferase activity (Supplemental Fig. 4, lane 3). This relatively simple experiment clearly demonstrates that proteins other than GfsA have no, or only very minimal, galactofuranosyltransferase activity.
We next attempted to determine if WscA-HA protein from strain 'gfsA can serve as an acceptor substrate for GfsA-3xFLAG protein. The galactofuranosyltransferase activity of purified GfsA protein was assayed in the presence of 0.25 g WscA-HA protein obtained from strain 'gfsA (Fig. 5E). Prior to its use an acceptor substrate, WscA-HA was treated with PNGase F to remove N-glycans. Analysis of the reaction products of the assay by immunoblotting with EB-A2 antibody showed that purified GfsA-3xFLAG protein had galactofuranosyltransferase activity in the presence of both WscA-HA and UDP-Galf (Fig. 5E, lane 1). In contrast, reaction mixtures without GfsA-3xFLAG, WscA-HA, or UDP-Galf did not have detectable enzymatic activity, as indicated by the absence of an EB-A2 staining signal (Fig. 5E, lanes 2, 3, and 4).
Biochemical analyses of GfsA from A. nidulans were also performed using the established in-vitro assay. EB-A2 signal intensity clearly increased in a time-dependent manner (Fig. 6A, lanes 1'5). GfsA was inactive toward galactomannoproteins from 'ugmA (GMP'ugmA) (Fig. 6B, lane 4), and was also relatively inactive in the presence of UDP-Galp, GDP-mannose, and UDP-glucose compared to UDP-Galf, which was associated with a strong EB-A2 signal (Fig. 6C, lanes 3-5). GfsA was also inactive in the presence of 10 mM EDTA, which can chelate Mn2+ (Fig. 6D, lane 2). Taken together, these results show that UDP-Galf and Mn2+ are required for GfsA activity.

Subcellular localization of GfsA protein - To determine the subcellular localization of GfsA in A. nidulans, we performed sucrose density gradient centrifugation with total subcellular fractions extracted from the A. nidulans strain expressing GfsA-3xFLAG (Fig. 7). The presence of GfsA and distribution of marker proteins were analyzed by immunoblotting with antibodies against UgtA/GlfB (a marker for the Golgi apparatus) and BipA (ER lumen). GfsA was predominantly detected in fractions 9 to 11. This pattern was similar to that observed for UgtA/GlfB, suggesting that GfsA colocalizes with UgtA/GlfB (Fig. 7). In contrast, BipA was detected at similar levels in fractions 1 to 9 (Fig. 7). Thus, the distribution pattern observed for GfsA indicates that GfsA is principally localized to the Golgi apparatus.

Abnormal cell morphology of strain 'gfsA - The disruption of gfsA inhibited hyphal extension and conidial formation. We observed that strain 'gfsA forms smaller colonies than wt after cultivation on minimal medium at 30C for 3 days (Fig. 8A). The colony growth rates of the wt and 'gfsA strains were 0.40 0.070 and 0.27 0.060 mm/h, respectively. The presence of an osmotic stabilizer (0.6 M KCl) slightly restored colony size in 'gfsA (Fig. 8A). Moreover, under high-temperature conditions (42C), the growth defects of strain 'gfsA were restored (Fig. 8A).
We also examined the conidiation efficiency of the 'gfsA and wt strains. Formation of normal green conidia was markedly repressed in 'gfsA grown on minimal medium at 30C for 3 days (Fig. 8A). The efficiency of conidiation in 'gfsA was reduced to approximately 11% of that of the wt strain, but recovered to as much as 86% of wt on minimal medium plates in the presence of 0.6 M KCl at 42C (Fig. 8B). Next, we tested the sensitivity of strain 'gfsA to SDS, Congo Red, and calcofluor white treatments (Fig. 8C). We found that the growth of strain 'gfsA was more sensitive than the wt strain to treatment with 0.002% (w/v) SDS (Fig. 8C), but was more resistant to 30 g ml-1 calcofluor white (Fig. 8C). Hyphal growth of the 'gfsA strain was not significantly affected by treatment with 30 g ml-1 Congo red (Fig. 8C).
Conidiophores grown on minimal medium plates at 30C for 3 days were next examined by scanning electron microscopy. Conidiophores of 'gfsA were sparse and scattered compared with those of wt (Fig. 9A (a) and (b)). In addition, the number of conidia per conidiophore was smaller in 'gfsA than in wt (Fig. 9A (c) and (d)). The structure of hyphae in liquid minimal medium was also investigated by fluorescence microscopy. The hyphae of wt A. nidulans grew linearly (Fig. 9B (a)), whereas strain 'gfsA formed curved hyphae with abnormal branching. The distance between septa in hyphae of strain 'gfsA was shorter than that in hyphae of wt (Fig. 9B (b)). Together, these observations suggest that gfsA disruption causes abnormal hyphal formation, but does not influence septum formation.

Transcriptional and protein expression analyses of gfsA - To elucidate the growth stage at which gfsA is expressed, we analyzed its transcription using real-time reverse transcription-PCR after 18, 24, 36, and 48 h of culture (Fig. 10A). The transcriptional levels of gfsA were relatively similar between 18 through 24 h, but markedly decreased between 24 and 48 h. We also assayed GfsA protein levels at 18, 24, 36 and 48 h (Fig. 10B). GfsA was expressed at high levels at 18 and 24 h, but the levels decreased after 24 h, whereas '-actin was expressed almost constitutively expressed (Fig. 10B). This result was comparable with the result of gfsA transcription analysis. In addition, to determine if gfsA is transcribed in conidia, we compared transcription of gfsA in conidia with that in mycelia. We first tested the quality of the conidia RNA sample by assaying levels of the conidia-specific spoC1-C1C transcript. The results indicated that the sample RNA was successfully extracted from conidia (Fig. 10C, right panel). Moreover, the level of gfsA RNA in the conidia was higher than that in the mycelia after cultivation in minimal liquid medium for 18 h (Fig. 10C, left panel). These results are consistent with the speculation that GfsA has a significant role not only during hyphal development, but also during conidiation and in the conidia.

Functional analyses of a gfsA ortholog in A. fumigatus - It has been suggested that Galf-containing molecules are related to virulence and infection of A. fumigatus, which is an opportunistic human pathogen (Schmalhorst, 2008; Lamarre, 2009). We identified a putative ortholog of gfsA (AFUB_096220) in A. fumigatus strain A1163. To analyze the function of this gene, which we termed AfgfsA, we generated an AfgfsA disruptant using A. fumigatus strains A1160 and A1151 as parental strains. Colonies formed by strain 'AfgfsA in the A1160 background were smaller than those formed by strain A1160 at 47C (Fig. 11A) and conidia formation was reduced. These phenotypes were restored in the presence of 0.6 M KCl at 47C. To confirm that the mutation could be complemented, we introduced the plasmid pPTR-II-gfsA into strain 'AfgfsA. The abnormal colony phenotype was restored in the 'AfgfsA strain expressing A. nidulans gfsA (Fig. 11B). In addition, Galf antigens in galactomannoproteins were detected by immunoblotting analysis with EB-A2. In the 'AfgfsA strain expressing gfsA from A. nidulans, the signal intensity of EB-A2 immunodetection was also recovered to wt levels, indicating that A. nidulans gfsA can synthesize the Galf antigen in both A. nidulans and A. fumigatus (Fig. 11C). This result also indicates that AfgfsA encodes a galactofuranosyltransferase.
Finally, we tested the sensitivity of A1151, 'glfA, and 'AfgfsA to micafungin and voriconazole on MEA medium. Plates with conidia covering the surface were test strips were incubated with test strips of the two antibiotics for 24 h at 37C. Under these conditions, A. fumigatus strain A1151 formed a clear zone of growth inhibition around voriconazole (minimum inhibitory concentration [MIC], 0.125 g ml-1) and micafungin (MIC, 0.008 g ml-1). However, strain 'AfgfsA showed a larger zone of growth inhibition around voriconazole (MIC, 0.064 g ml-1l) and micafungin (MIC, 0.004 g ml-1), similar to that formed by strain 'glfA strain (Fig. 11D), indicating that 'AfgfsA has increased sensitivity to antifungal agents.

Discussion
Strain 'ugmA displays an abnormal phenotype that appears to result from the loss of all Galf-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, galactofuran side chains, and glycosylinositolphosphoceramides. To help clarify the roles of individual Galf-containing polysaccharides and glycoconjugates, it is necessary to identify and characterize genes that encode galactofuranosyltransferases involved in the synthesis of these molecules. Towards this goal, here, we attempted to identify a galactofuranosyltransferase gene involved in Galf antigen synthesis in the filamentous fungus A. nidulans. Using reverse-genetic and biochemical approaches, we provide evidence that gfsA encodes a galactofuranosyltransferase with the ability to synthesize the Galf antigen of O-glycans. To our knowledge, this is the first report describing a galactofuranosyltransferase-encoding gene that functions in the fungal Galf synthetic pathway. Engel and colleagues proposed a novel schematic model of galactofuranosylation (Engel, 2009). Based on our present results, we have updated the model of galactofuranosylation in fungal cells to include GfsA protein (Fig. 12). In the updated model, UDP-Galf is first synthesized from UDP-glucose via UDP-Galp in the cytosol by UgeA and UgmA/GlfA (Damveld, 2008; Schmalhorst, 2008; El-Ganiny, 2008; Lamarre, 2009; El-Ganiny, 2010) and is then transported into the Golgi apparatus via the antiporter protein UgtA/GlfB (Engel, 2009; Afroz, 2011). GfsA then synthesizes Galf antigen of O-glycans on galactomannoproteins in the Golgi apparatus during hyphal tip growth. Finally, the synthesized galactofuranosylated proteins are transported by vesicles and localized to the cell surface (Fig. 12).

The reactivity of monoclonal antibody EB-A2 with Galf antigen present on galactomannoproteins was reduced in strain 'gfsA, suggesting that gfsA is involved in the synthesis of Galf antigen (Fig. 2B). EB-A2 reactivity towards purified WscA protein, an O-glycosylated protein, was also reduced following disruption of gfsA (Fig. 4D). In addition, the results of in-vitro assays provide evidence that purified GfsA protein has Galf antigen synthase activity toward WscA protein and GMP'gfsA, using UDP-Galf as a sugar donor (Fig. 5D and E). However, GfsA does not show activity toward GMP'ugmA (Fig. 6B, lane 4). Based on the structure of Galf antigen in O-glycans (Fig. 1), at least two galactofuranosyltransferases appear to be involved in the synthesis of Galf antigen; one forms a '1,6-linkage between Galf and the '1,6-mannobiose backbone, and the other forms '1,5-Galf linkages between Galf residues at the non-reducing termini. As the latter reaction requires a Galf residue as an acceptor substrate for catalysis, galactomannnoproteins extracted from 'ugmA strain cannot serve as a substrate for this enzyme. Therefore, it is highly likely that GfsA is a novel '1,5-galactofuranosyltransferase involved in O-glycan biosynthesis. However, details studies of the type of linkage for the Galf residue and the substrate structure of GfsA are warranted.

A. nidulans strains 'ugmA and 'ugtA exhibit defects in colony growth, hyphal morphogenesis, and conidiation (El-Ganiny, 2008; Afroz, 2011). Strain 'gfsA exhibits defects similar to those of 'ugmA (Fig. 1A), including abnormal cell morphology, poor hyphal extension, hyphal curvature, and limited conidia formation (Figs. 8A, 8B, 9A and 9B). Similar phenotypes were also observed in strains 'ugmA and 'pmtA, indicating that Galf residues in O-mannose type glycans are required for normal cell morphology (Oka, 2004; El-Ganiny, 2008). If GfsA is also a galactosyltransferase involved in the synthesis of galactofuran side chains, our findings also indicate that these side chains are also required for normal cell morphology.

Genes involved in the Galf biosynthetic pathway are thought to be functionally linked to cell wall formation and maintenance (El-Ganiny, 2008; El-Ganiny, 2010; Afroz, 2011). The growth defect of strain 'gfsA was not observed under conditions of high temperature and hyperosmolarity, a finding that is also reported for mutants with defects in cell wall integrity (Horiuchi, 1999; Oka, 2004; Goto, 2009; Futagami, 2011). The molecular mechanisms that control the ability of Aspergillus spp. to maintain cell wall integrity under stress have not been fully elucidated. Nevertheless, it is reasonable to speculate that in strain 'gfsA, the triggering of stress responses, such as those normally induced by heat and hyperosmotic stress, might help to compensate for the loss of Galf antigen in cell wall O-glycans. Strain 'gfsA shows sensitivity to Congo red (30 g ml-1), an inhibitor of glucan synthesis, but is resistant to calcofluor white (30 g ml-1), an inhibitor of chitin synthesis. These results imply that the balance of cell wall components is altered in strain 'gfsA, perhaps due to the reduction in Galf antigen and/or an increase in chitin levels. El-Ganiny (2008) reported that the abnormal hyphal phenotype that is observed in the A. nidulans 'ugmA strain under normal growth conditions can be partially suppressed by growth on medium containing 10 g ml-1, but not 30 g ml-1, calcofluor white. This finding is different from that observed for strain 'gfsA, suggesting that the function of the Galf antigen of O-glycans differs from those of other antigens, including the galactofuran side chain, N-glycan, and glycosylinositolphosphoceramide, at least as they relate to resistance or sensitivity to calcofluor white.

Disruption of ugmA leads to a complete loss of galactofuranose residues in the cell, because UDP-Galf, which is the sugar donor for all galactofuranosyltransferases, is not synthesized. In contrast, galactofuranose residues were detected in strain 'gfsA (Fig. 2C), providing further evidence that GfsA protein is a galactofuranosyltransferase that can transfer galactofuranose from UDP-Galf to O-glycans. Consistent with this speculation, the defects observed in strain 'gfsA were less severe than those observed for 'ugmA (Fig. 2A), demonstrating that other galactofuranose residues, in addition to O-glycans, are also important for normal cell growth.

We also found that galactomannnoproteins extracted from 'gfsA are weakly recognized by EB-A2, even though no signal was detected with proteins from 'ugmA (Fig. 3D, lane 2; Fig. 4C, lane 5). A signal was seen for WscA purified from the 'gfsA mutant because a larger number of WscA molecules were present on the gel compared to individual galactomannoproteins (Fig. 4C, lane 5). This observation indicates that galactofuranosyltransferase activity remained in the 'gfsA cells. However, galactofuranosyltransferase activity was not detected in the solubilized protein fraction extracted from 'ugmA'gfsA (Supplemental Fig. 4, lane 3). Although these results are at first contradictory, they suggest that GfsA plays a major role in the transfer of galactofuranose residue to O-glycans in cells and that the conditions of our in-vitro assay were not suitable to detect the remaining galactofuranose activity in strain 'ugmA.

The gfsA gene was identified among candidate genes selected from the CAZy database and belongs to the GT31 family, which includes galactosyltransferases such as glycoprotein-N-acetylgalactosamine-''''-galactosyltransferase (beta3GalT1) (Bardoni, 1999), hydroxyproline-O-galactosyltransferase (Basu, 2013), and glycosaminoglycan-1,3-galactosyltransferase (Zhou, 1999). In the A. nidulans genome, there are seven genes encoding GT31 family proteins (AN2015, AN4824, AN5663, AN7535, AN8627, AN11144, and AN11697) in addition to gfsA. Of these genes, AN2015 and AN5663 appear to be the most closely related to gfsA. Thus, it is likely that these genes are also involved in the addition of Galf residues to O-glycans. These genes will be examined in future experiments in an effort to identify additional galactofuranosyltransferases involved in the synthesis of Galf-containing polysaccharides and glycoconjugates.

Fungal-type galactofuran side chains of galactomannan are '1,5-linked Galf oligomers composed of up to five residues. Galactofuran side chains are linked to '1,2-mannan by '1,6- or '1,3-linkages (Latg, 1994). In yeast, the mannosyltransferase Mnn1 is responsible for the synthesis of both O-glycans and the outer chain of N-glycans (Lussier, 1994). Because the structure of galactofuran side chains is similar to the structure of O-glycans (Fig. 1), we cannot exclude the possibility that GfsA, and/or its putative paralogs, have the ability to biosynthesize fungal-type galactofuran side chains of galactomannan.

Genes related to gfsA are widely distributed in the subphylum Pezizomycotina of Ascomycota, but are not found in the subphyla Saccharomycotina or Taphrinomycotina (Table S3). In contrast, basidiomycete fungi, including Cryptococcus neoformans and Ustilago maydis, have both ugmA/glfA and ugtA/glfB orthologs, but no ortholog of gfsA (Table S3) (Beverley, 2005). Consistent with this finding, no binding of EB-A2 to exopolysaccharides from C. neoformans, Candida albicans, Saccharomyces cerevisiae or Rhizopus stolonifer was observed (Stynen, 1992; Kappe, 1993). To our knowledge, Galf antigen structures are not found in C. neoformans, despite the fact that a non-reducing terminal Galf residue is observed in galactoxylomannans from C. neoformans (James, 1992). These observations are consistent with C. neoformans having both ugmA and ugtA/glfB orthologs, but no gfsA ortholog. In the phylum Zygomycota, which includes fungi such as Rhizopus oryzae RA 99-880, no genes involved in Galf synthesis have been detected, including gfsA orthologs (Table S3). It seems likely that fungal species containing the gfsA gene would also have both ugmA/glfA and ugtA/glfB, as the products of these genes act upstream of GfaA in the Galf synthesis pathway.

The Pezizomycotina subphylum contains fungi pathogenic to humans, animals, and plants. It has been suggested that Galf-containing polysaccharides and glycoconjugates from these fungi are related to virulence and infection (Pedersen, 2003; Latg, 2009; Tefsen, 2012). Moreover, in the present study, we found that strain 'AfgfsA of the opportunistic human pathogen A. fumigatus exhibits a small-colony phenotype and has increased sensitivity to antifungal treatments. These characteristics suggest that GfsA activity might be an appropriate new target for antifungal treatment (Fig. 11A and D). We anticipate that our findings will stimulate functional investigation into the medical and biotechnological implications of Galf-containing polysaccharides and glycoconjugates.
Experimental procedures
Microorganisms and growth conditions - The A. nidulans and A. fumigatus strains used in this study are listed in Table S2. A. nidulans strain AKU89 was previously described (Goto, 2009). A. fumigatus A1160 and A1151 (da Silva Ferreira, 2006) were obtained from the Fungal Genetics Stock Center (FGSC). Strains were grown on minimal medium (1% (w/v) glucose, 0.6% (w/v) NaNO3, 0.052% (w/v) KCl, 0.052% (w/v) MgSO4'7H2O, 0.152% (w/v) KH2PO4, biotin (trace), and Hunter's trace elements, pH 6.5) (Barratt, 1965), complete medium (CM) with appropriate supplements for nutritional markers as described previously (Kaminskyj, 2001), YG medium (0.5% yeast extract and 1% glucose) or MEA medium (2% malt extract, 2% glucose, 0.1% peptone, and 2% agar, pH 6.0). Growth experiments to allow hyphal development in submerged culture were begun by inoculation of 100 ml minimal medium with 2 107 conidia in 500-ml culture flasks. The flasks were shaken at 126 rpm at 30C for A. nidulans and 37C for A. fumigatus. Standard transformation procedures for Aspergillus strains were used (Yelton, 1984). Plasmids were amplified in Escherichia coli JM109 or XL1-Blue.

Construction of deletion cassettes and gene disruption strains - A. nidulans AKU89, A. fumigatus A1160, and A. fumigatus A1151 were used as wild-type (wt) control strains (Table S2). The pyrG gene was amplified by PCR using A. nidulans FGSC26 genomic DNA as template and primers AnpyrG-F and AnpyrG-R (Table S4). The amplified fragments were inserted into the EcoRV sites of pGEM-5Zf(+) using the pGEM-T Easy Vector System (Promega Co., Madison, WI) to yield pSH1. Genes were disrupted in A. nidulans by argB or ptrA insertion. AfgfsA was disrupted in strain A1160 by pyrG insertion, and glfA and AfgfsA were disrupted in strain A1151 by ptrA insertion. DNA fragments for gene disruption were constructed using a 'double-joint' PCR method, as described previously (Yu, 2004). All PCR was performed using Phusion High-Fidelity DNA Polymerase (Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan). Primers used in this study are listed in Table S4. Eight primers (from ANxxxx-1 to ANxxxx-8 for A. nidulans genes or from AFUB_xxxxxx-1 to AFUB_xxxxxx-8 for A. fumigatus genes; xxxx or xxxxxx indicates the systematic gene name) were used to construct deletion cassettes. The 5'- and 3'-flanking regions (approximately 0.9-1.2 kb each) of each gene were PCR amplified from genomic DNA with primer pairs ANxxxx-1/ANxxxx-2 or AFUB_xxxxxx-1/AFUB_xxxxxx-2 and ANxxxx-3/ANxxxx-4 or AFUB_xxxxxx-3/ AFUB_xxxxxx-4, respectively (Table S4). The argB, pyrG, and ptrA genes used as selective markers were amplified using plasmids pDC1 (Aramayo, 1989, obtained from the FGSC), pSH1, and pPTR-I (Takara Bio, Inc., Otsu, Japan), respectively, as template and the primer pairs ANxxxx-5/ANxxxx-6 or AFUB_xxxxxx-5/AFUB_xxxxxx-6. The three amplified fragments were purified and mixed and a second PCR was performed without specific primers, as the overhanging chimeric extensions act as primers, to assemble each fragment. A third PCR was performed with the nested primer pairs ANxxxx-7/ANxxxx-8 or AFUB_xxxxxx-7/AFUB_xxxxxx-8 and the products of the second PCR as template to generate the final deletion construct. The amplified final deletion constructs were purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and used directly for transformation. Transformants were grown on minimal medium plates containing 0.6 M KCl as an osmotic stabilizer under appropriate selection conditions and single colonies were isolated twice before further analysis.
Disruption of target genes was confirmed by PCR with the primer pairs ANxxxx-1/argB-R and argB-F/ANxxxx-4 or ANxxxx-1/ptrA-R and ptrA-F/ANxxxx-4 for A. nidulans, AFUB_xxxxxx-1/pyrG-R and pyrG-F/AFUB_xxxxxx-4 for A. fumigatus A1160, and AFUB_xxxxxx-1/ptrA-R and ptrA-F/AFUB_xxxxxx-4 for A. fumigatus A1151. Disruption of gfsA in transformants was confirmed by Southern blotting. The 5'-UTR of gfsA was amplified by PCR with the primer pair gfsA-probe-F/gfsA-probe-R. The amplified fragment was labeled with digoxigenin and then used as a probe.
To construct an argB+ derivative of the A. nidulans parent strain AKU89, the entire argB gene was amplified from genomic DNA of A. nidulans FGSC26 by PCR with the primer pair argB-RESCUE-F and argB-RESCUE-R (Table S4). The 2.5-kb amplified fragment was purified using a QIAquick Gel Extraction Kit (Qiagen) and used directly for transformation to generate strain AKUARG89.

Construction of strains 'gfsA-AnwscA and 'ugmA-AnwscA - A. nidulans AKU89-AnwscA (Goto, 2009) was used as a parental strain for the construction of strains 'gfsA-AnwscA and 'ugmA-AnwscA (Table S2). The gfsA and ugmA genes were disrupted in AKU89-AnwscA by ptrA insertion. Eight primers (from AN8677-1 (gfsA::ptrA) to AN8677-8 (gfsA::ptrA) or AN3112-1 (ugmA::ptrA) to AN3112-8 (ugmA::ptrA)) were used to construct a deletion cassette. DNA fragments for gene disruption were constructed using a double-joint PCR method (described above). Disruption of gfsA and ugmA was confirmed by PCR with the primer pairs AN8677-1 (gfsA::ptrA)/ptrA-R and ptrA-F/AN8677-4 (gfsA::ptrA) or AN3112-1 (ugmA::ptrA)/ptrA-R and ptrA-F/AN3112-4 (ugmA::ptrA).

Purification of WscA-HA protein ' HA-tagged WscA was purified from strains AKU89-AnwscA, 'gfsA-AnwscA, and 'ugmA-AnwscA that were grown in 100 ml minimal medium at 30C for 24 h and harvested by filtration. The cells (1 g wet cells) were ground in liquid nitrogen with a mortar and pestle, and the lysed cells were resuspended in buffer B (50 mM HEPES-NaOH (pH 6.8), 100 mM NaCl, 30 mM KCl, and 1% Triton X-100). After cell debris was removed by centrifugation at 3,000 x g for 10 min, 20 l of rabbit polyclonal anti-HA-Tag-agarose (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) was added to the supernatant and the mixture was gently shaken for 3 h. The anti-HA-Tag-agarose was collected by centrifugation at 1,400 x g for 10 min, and was then washed five times with 1.5 ml buffer B.

Amplification, cloning, and sequencing of gfsA cDNA - The gfsA cDNA was amplified by PCR using a 24-h developmental cDNA library from A. nidulans that was constructed by R. Aramayo (obtained from the FGSC) as a template and the primers AN8677-cDNA-F/AN8677-cDNA-R (Table S4). The amplified cDNA fragments were inserted into the EcoRV sites in pGEM-5Zf(+) using the pGEM-T Easy Vector Systems (Promega Co.) to yield pTOGFSA. The sequence of gfsA was confirmed using an Applied Biosystems 3130xl Genetic Analyzer (Life Technologies Co., Carlsbad, CA).

Extraction of galactomannoproteins - Galactomannoproteins were extracted according to the method described by Chiba (2008). Aspergillus cells were grown in minimal medium at 30C or 37C for 24 h. The cells were harvested by filtration, washed twice with distilled water, resuspended in 100 mM citrate buffer (pH 7.0), and then autoclaved at 121C for 120 min. After cell debris was removed by filtration, the extracted galactomannoproteins were precipitated by ethanol, dialyzed with dH2O, and then lyophilized. The protein concentration of the purified galactomannoproteins was measured based on absorbance at 280 nm (A280).

Determination of D-galactopyranose content of galactomannoproteins - The extracted galactomannoproteins (2.4 g) were lyophilized, incubated in 4 M TFA at 100C for 4 h, and then dried at room temperature. The hydrolysates were labeled with fluorescent p-aminobenzoic acid ethyl ester (ABEE) using an ABEE Labeling Kit (J-Oil Mills, Inc., Tokyo, Japan) according to the manufacturer's protocol. Protein levels were measured using a Qubit protein assay kit (Life Technologies Co.). ABEE labeled D-galactopyranose was analyzed using a high-performance liquid chromatography system equipped with a Honenpak C18 column (4.6 mm x 75 mm) (J-Oil Mills, Inc.). D-galactose (Sigma Aldrich, St. Louis, MO, USA) was used as a standard for quantification.

Release of O- and N-glycans from extracted galactomannoproteins - To release N-glycan, galactomannoproteins (72 g) were treated with PNGase F (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. To release O-glycan from cell wall proteins, '-elimination was performed using the GlycoProfile Beta-Elimination Kit (Sigma-Aldrich) according to the manufacturer's protocol. The '-elimination reaction was performed at 25C for 24 h.

Construction of an expression vector for gfsA - pPTR-II (Takara Bio, Inc.), which is an autonomously replicating plasmid containing the AMA1 sequence (Kubodera, 2002), was used an expression vector for gfsA. The gfsA gene was amplified by PCR using A. nidulans genomic DNA as template and the primers gfsA-In-fusion-F and gfsA-In-fusion-R. The amplified fragments were inserted into the SmaI sites of pPTR-II using an In-FusionTM Advantage PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA) to yield pPTR-II-gfsA. A. nidulans strain 'gfsA was transformed with pPTR-II-gfsA or pPTR-II. Transformants were selected on minimal medium supplemented with 0.1 mg ml-1 pyrithiamine and 0.6 M KCl. For each construct, three different transformants were randomly selected for subsequent experiments.

Construction of GfsA-3xFLAG-expressing strain - All PCR was performed using Phusion High-Fidelity DNA Polymerase (Daiichi Pure Chemicals Co., Ltd). A 3xFLAG-tagged DNA fragment was synthesized by PCR without template using the primer pair 3xFLAG-F/3xFLAG-R. The amplified fragments were inserted into the SphI sites of pDC1 to yield pDC1-3xFLAG. The open reading frame and upstream promoter of gfsA were amplified from genomic DNA by PCR with the primer pair AN8677-3xFLAG-1/AN8677-3xFLAG-2. Similarly, the terminator regions of the gfsA gene were amplified with the primer pair AN8677-3xFLAG-3/AN8677-3xFLAG-4. The argB gene, including the 3xFLAG tag, was amplified with the primer pair AN8677-3xFLAG-5/AN8677-3xFLAG-6 using plasmid pDC1-3xFLAG as template. The three DNA fragments were mixed and a second PCR was performed with the nested primers AN8677-3xFLAG-7/AN8677-3xFLAG-8 to generate the final DNA construct for chromosomal tagging. The final amplified deletion constructs were purified using a QIAquick Gel Extraction Kit (Qiagen) and used directly for transformation.

Construction of strain GfsA-3xFLAG ('ugmA) - AThe ugmA gene was disrupted in A. nidulans GfsA-3xFLAG (Table S2) by ptrA insertion. Eight primers (from AN3112-1 (ugmA::ptrA) to AN3112-8 (ugmA::ptrA)) were used to construct a deletion cassette. DNA fragments for gene disruption were constructed using the double-joint PCR method (described above). Disruption of ugmA was confirmed by PCR with the primer pairs AN3112-1 (ugmA::ptrA)/ptrA-R and ptrA-F/AN3112-4 (ugmA::ptrA).

Preparation of solubilized protein - Cells from GfsA-3xFLAG ('ugmA), 'ugmA or 'ugmA'gfsA were collected by centrifugation and were then ground in liquid nitrogen with a mortar and pestle. The lysed cells were resuspended in buffer A (50 mM HEPES-NaOH (pH 6.8), 100 mM NaCl, 30 mM KCl, 1 mM MnCl2, and 5% glycerol (w/v)). All procedures were performed on ice or at 4C. Cell debris was removed by centrifugation at 10,000 x g for 10 min, and the obtained supernatants were further centrifuged at 100,000 x g for 30 min. The resultant pellet was resuspended in buffer A with 0.2% CHAPSO (Dojindo Laboratories, Kumamoto, Japan). To obtain solubilized membrane proteins, the sample was centrifuged at 100,000 x g for 30 min after stirring for 1 h.

Purification of GfsA-3xFLAG protein - GfsA protein purified using anti-FLAG beads from strain GfsA-3xFLAG ('ugmA) was used as a source of enzyme. A protein sample purified from strain 'ugmA using the same procedure was used as a negative control. The strains GfsA-3xFLAG ('ugmA) and 'ugmA were grown in 2 l minimal medium at 30C for 24 h and then harvested by filtration. The solubilized proteins were prepared from 25 g of wet cells as described above. Mouse-IgG-agarose (100 l; Sigma-Aldrich) was added to the supernatant and the mixture was gently shaken for 1 h. Mouse-IgG-agarose was removed by centrifugation at 1,400 x g for 10 min. A total of 200 l anti-FLAG M2 affinity gel (Sigma-Aldrich) was then added to the supernatant and the resultant mixture was gently shaken for 1 h. Anti-FLAG M2 affinity gel was collected by centrifugation at 1,400 x g for 10 min and then washed five times with 50 ml buffer A containing 0.2% CHAPSO. GfsA protein was then eluted with 200 l buffer A with 0.2% CHAPSO containing 0.5 g l-1 3xFLAG peptide (Sigma-Aldrich). The eluted proteins obtained from strains GfsA-3xFLAG ('ugmA) and 'ugmA were designated GfsA-3xFLAG and control, respectively. The protein concentration was determined using the bicinchoninic acid protein assay reagent (Thermo Fisher Scientific, Waltham, MA) with bovine serum albumin as a standard.

In-vitro assay of galactofuranose antigen synthase activity - UDP-Galf was synthesized and then purified by HPLC, as previously described (Oppenheimer, 2010). UDP-Galf was used as a sugar donor and purified WscA-HA, galactomannoproteins from 'gfsA (GMP'gfsA), or galactomannoproteins from 'ugmA (GMP'ugmA) were used as acceptor substrates. Galf antigen synthase was assayed in a reaction mixture containing 25 mM HEPES-NaOH (pH 6.8), 50 mM NaCl, 15 mM KCl, 0.5 mM MnCl2, 2.5% glycerol (w/v), 0.72 mM UDP-Galf acceptor substrate, and 14 g purified GfsA-3xFLAG protein in a total volume of 10 l. For the assay, 2.5 g GMP'gfsA, GMP'ugmA or 0.25 g purified WscA-HA was used as an acceptor substrate. The mixture was incubated at 37C and the reaction was stopped by incubation at 99C for 5 min. The reaction products were analyzed by immunoblotting (described below).

Antibodies - The following antibodies were used for immunoblotting. Anti-BipA antibodies were described previously (Goto, 2004). Anti-FLAG M2 and anti-UgtA/GlfB antibodies were purchased from Sigma-Aldrich. Anti-UgtA/GlfB antibody was raised by subcutaneous immunization of a rabbit with a synthetic peptide (CEPTLPTVNPAVDKPEPPK) conjugated to KLH, and the serum was tested using an enzyme-linked immunosorbent assay. Anti-FLAG M2 mouse monoclonal antibody (1:5000; Sigma-Aldrich), anti-HA F-7 mouse monoclonal antibody (1:5000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-'-actin monoclonal antibody clone C4 (1:200; MP Biomedicals, LLC., Santa Ana, CA), UgtA/GlfB (1:1000), and BipA (1:200) were used as primary antibodies. Anti-mouse IgG conjugate HRP (Santa Cruz Biotechnology, Inc.) was used to detect anti-FLAG M2; anti-'-actin and anti-UgtA/GlfB was used to detect UgtA/GlfB; and anti-rabbit IgG conjugate HRP (Santa Cruz Biotechnology, Inc.) was to detect anti-BipA. EB-A2 antibody, which is one of the components of Platelia Aspergillus EIA (Bio-Rad Laboratories, Hercules, CA), was used at a dilution of 1:10 to detect '-Galf antigen. All secondary antibodies were used at a dilution of 1:5000.

Immunoblotting analysis - Galactomannoproteins were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane using an AE-6677 electroblotter (Atto Co., Tokyo, Japan) at 100 mA for 1.5 h. After incubation of the membrane for 1 h in a blocking buffer containing 10 mM phosphate (pH 7.4), 4% skim milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 0.1% Tween 20, and 0.9% (w/v) NaCl, the membrane was transferred to a 5-ml solution of primary antibody. The membrane was incubated for 12 h at 4C, washed three times with a buffer containing 10 mM phosphate (pH 7.4), 0.1% Tween 20, and 0.9% (w/v) NaCl for a total of 30 min, and then incubated for 1 h with secondary antibody. An ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) was used to visualize the immunoreactive proteins. Chemical fluorescent signals on the membrane were recorded using a MicroChemi imaging system (Berthold Technologies, Bad Wildbad, Germany). Imaging of the ECL-treated membranes was stopped before saturation of the signals.

Colony growth rate determination - Colony growth rates were measured as described previously (Kellner, 2002). Briefly, conidia from each strain were point-inoculated into the center of agar medium plates. Colony diameters were measured after 24, 48, 72, 96, and 120 h of incubation at 30C. The growth rates were determined for each colony in millimeters per hour during each of the incubation intervals, i.e., 24'48, 48'72, 72'96, and 96'120 h, and were then averaged across the entire time interval. Measurements of growth rates for all individual strains were performed 20 times.

Analysis of the efficiency of conidiation - The efficiency of conidiation was analyzed as described previously (Oka, 2004). Briefly, approximately 105 conidia were spread onto a minimal medium plate (90-mm diameter). After 5 days of incubation at 30C, the conidia were suspended in 5 ml of 0.01% (w/v) Tween 20 and counted using a hemocytometer.

Microscopy - Hyphae cultured in liquid minimal medium were observed using a Fluoview FV10i confocal laser-scanning microscope (Olympus Co., Tokyo, Japan). The mycelia were harvested and incubated with 10 ng ml-1 Fluorescent Brightener 28 (calcofluor white; Sigma-Aldrich) for 10 min. Conidiophores were observed using a VE-8800 scanning electron microscope (Keyence, Osaka, Japan).

Transcription analysis of gfsA - For RNA extraction from mycelia, conidia (2 107) of A. nidulans wt strain (AKUARG89) were inoculated into 100 ml liquid minimal medium and were then grown for 18, 24, 36, and 48 h at 30C. For RNA extraction, approximately 2 x 105 conidia were spread onto an 84-mm agar plate of minimal medium. After a 3-d incubation at 30C, conidia were collected and then ground for 90 sec at 2,000 rpm with a Multi-Beads Shocker (Yasui Kikai Co., Osaka, Japan). RNA was extracted using RNAiso PLUS (Takara Bio, Inc.) according to the manufacturer's protocol and was then quantified using a Quant-iT RNA Assay Kit (Life Technologies Co.). cDNA was synthesized from the RNA using a PrimeScript Perfect Real-Time Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. Real-time reverse transcription (RT)-PCR analysis was performed using a LightCycler Quick 330 system (Roche Diagnostics) with SYBR Premix DimerEraser (Perfect Real Time; Takara Bio, Inc.). The following primers were used: gfsA-RT-F and gfsA-RT-R for gfsA, histone-RT-F and histone-RT-R for the histone H2B, argB-RT-F and argB-RT-R for argB, and spoC1-C1C-RT-F and spoC1-C1C-RT-R for spoC1-C1C (Table S4). The histone H2B gene was used to standardize the mRNA levels of the target genes (Fujioka, 2007).

Organelle separation - Organelles were separated on sucrose gradients as described previously (Engel, 2009). Briefly, A. nidulans cells were grown in minimal medium at 30C for 24 h. All manipulations were performed on ice or at 4C. Cells were harvested and ground in liquid nitrogen with a mortar and pestle. The lysed cells were suspended at 0.5 ml g-1 in 50 mM MOPS-NaOH, pH 7.0. Cell debris was removed by centrifugation at 3,000 x g. A discontinuous gradient was formed by sequentially adding 2.4 mL each of 55% sucrose, 50% sucrose, 45% sucrose, 40% sucrose, and 35% sucrose. A total of 1.7 mL of supernatant was then loaded onto the upper 35% sucrose solution, and the tube was centrifuged at 100,000 x g for 18 h, and a total of 16 fractions were collected. Specific membrane fractions were identified by immunoblotting using antibodies against organelle-specific markers.

Sensitivity testing ' To assay the sensitivity of A. fumigatus to antifungal treatment, Etests for micafungin and voriconazole was performed using the appropriate test strips (Sysmex bioMrieux Co., Ltd., Lyon, France) on MEA plates. For the assay, conidia were harvested, washed twice with distilled water, and then counted using a hemocytometer. Conidia (6 x 105) were then evenly spread and test strips were placed in the center of the plate before incubation at 37C for 24 h.

Software and database searches ' The structure of GfsA was analyzed using the SOSUI tool (http://bp.nuap.nagoya-u.ac.jp/sosui/) (Hirokawa, 1998). For similarity searches, we used the Multi-fungi BLAST program (http://www.broadinstitute.org/annotation/genome/FGI_Blast/Blast.html) and the AspGD Multi-Genome Search using NCBI BLAST+ (http://www.aspgd.org/cgi-bin/compute/blast_clade.pl) (Altschul, 1997).

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