Identification of galactofuranose antigens such as galactomannoproteins and fungal-type galactomannan from the yellow koji fungus (Aspergillus oryzae)

Abstract

Filamentous fungi belonging to the genus Aspergillus are known to possess galactomannan in their cell walls. Galactomannan is highly antigenic to humans and has been reported to be involved in the pathogenicity of pathogenic filamentous fungi, such as A. fumigatus, and in immune responses. In this study, we aimed to confirm the presence of D-galactofuranose-containing glycans and to clarify the biosynthesis of D-galactofuranose-containing glycans in Aspergillus oryzae, a yellow koji fungus. We found that the galactofuranose antigen is also present in A. oryzae. Deletion of ugmA, which encodes UDP-galactopyranose mutase in A. oryzae, suppressed mycelial elongation, suggesting that D-galactofuranose-containing glycans play an important role in cell wall integrity in A. oryzae. Proton nuclear magnetic resonance spectrometry revealed that the galactofuranose-containing sugar chain was deficient and that core mannan backbone structures were present in ΔugmA A. oryzae, indicating the presence of fungal-type galactomannan in the cell wall fraction of A. oryzae. The findings of this study provide new insights into the cell wall structure of A. oryzae, which is essential for the production of fermented foods in Japan.

Keywords: Aspergillus oryzae; UDP-galactopyranose mutase (UGM); cell wall; fungal-type galactomannan; galactofuranose.

Figure 1

Immunoblot analysis of galactomannoproteins from Aspergillus spp. The presence of Galf-containing glycoproteins was detected using EB-A2. Lanes 1–5: 20 μg of galactomannoproteins from Aspergillus fumigatus A1151 (Lane 1), Aspergillus nidulans A26 (Lane 2), Aspergillus oryzae RIB40 (Lane 3), Aspergillus luchuensis (Lane 4), and Aspergillus kawachii IFO4308 (Lane 5) were loaded. Densitometric quantification of immunoblotting bands was performed using ImageJ software (Schneider et al., 2012). The intensity ratios of EB-A2 for galactomannoproteins were calculated, and the ratio for A. fumigatus A1151 was normalized to 1.0.

Figure 2

Phylogenetic analysis of UDP-galactopyranose mutase family proteins from bacteria, Basidiomycota, and Pezizomycotina species. Protein sequences were downloaded from NCBI. The phylogenetic tree was drawn using iTOL, and the alignment and phylogenetic tree inference were performed using MAFFT and RAxML, included in ETE v3. The UgmA homologous protein from the bacteria Fibrella rubiginis, presumably with a common ancestor, was used as the outgroup.

Figure 3

Phenotypic analysis of the ΔugmA strain. (A) Colonial morphology of parental (NSPlD1) and ΔugmA strains on minimal medium (MM) agar and MM agar supplemented with 0.6 M KCl after culture at 30°C, 37°C, or 42°C for 4 days. Agar medium was inoculated with 1.0 × 10⁴ conidiospores. (B) Colonial morphology of the parental, ΔugmA, and ΔugmA + A. oryzae ugmA strains after culture on MM agar for 4 days. Agar medium was inoculated with 1.0 × 10⁴ conidiospores. (C) Sensitivity to the cell wall stress inducer calcofluor white (CFW). Parental and ΔugmA strains were grown on MM agar supplemented with 10 or 30 μg/ml CFW at 30°C for 3 days.

Figure 4

Morphology of the parental and ΔugmA strains. (A) Morphology of the hyphae of the parental and ΔugmA strains. (B) Hydrophobicity of the hyphae of the parental and ΔugmA strains. Hydrophobicity was indicated by the adherence of latex beads to the hyphae.

Figure 5

Expression of Aspergillus oryzae ugmA complements the A. nidulans ΔAnugmA phenotype. (A) Colonial morphology of AKU89A (control), ΔAnugmA, and ΔAnugmA + A. oryzae ugmA strains after culture on MM agar at 30°C for 4 days. Agar medium was inoculated with 1.0 × 10⁴ conidiospores. (B) Immunoblot analysis of galactomannoproteins using EB-A2. Lanes 1–3: 20 μg of galactomannoproteins extracted from AKU89A (Lane 1), ΔAnugmA (Lane 2), and ΔAnugmA + A. oryzae ugmA (Lane 3) were loaded.

Figure 6

Proton nuclear magnetic resonance (¹H-NMR) spectrometry of fungal-type galactomannan (FTGM) fraction from Aspergillus oryzae. The signal at 5.195 and 5.05 ppm of the ¹H-NMR spectra is the H-1 signal of the C-1 position of the underlined Galf residue in the β-Galf-(1,5)-β-Galf-(1,5)-β-Galf and β-Galf-(1,5)-β-Galf-(1,6)-β-Galf structures (Kudoh et al., 2015). Signals A (5.104 ppm), B (5.233 ppm), C (5.216 ppm), and D (5.054 ppm) from the ¹ H-NMR spectra were derived from H-1 at the C-1 position of the underlined D-Mannose (Man) residues in the structures -(1,6)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,6)- (A), −(1,6)-α-Man-(1,2)- α-Man-(1,2)- α-Man-(1,2)-α-Man-(1,6)- (B), −(1,6)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,6)- (C), and -(1,6)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,2)-α-Man-(1,6)- (D). The proton chemical shifts were referenced relative to internal acetone at δ 2.225 ppm.

Figure 7

Predicted model of the fungal-type galactomannan (FTGM) biosynthetic pathway in Aspergillus oryzae. Homologs of each FTGM biosynthesis-related protein in A. oryzae were identified by BLASTP at NCBI using Uge5 (Afu5g10780/AFUB_058380), GlfB (Afu3g12700/AFUB_036470), GfsA (Afu6g02120/AFUB_096220), GmtA (Afu5g05740/AFUB_053290), CmsA (Afu5g02740/AFUB_051270), CmsB (Afu5g12160/AFUB_059750), and AnpA (Afu4g06870/AFUB_063940) of A. fumigatus as queries.