Characterization of Cell Wall α-1,3-Glucan-Deficient Mutants in Aspergillus oryzae Isolated by a Screening Method Based on Their Sensitivities to Congo Red or Lysing Enzymes.

We previously reported that sensitivity to Congo Red (CR) or Lysing Enzymes (LE) is affected by the loss of cell-wall α-1,3-glucan (AG) in Aspergillus nidulans. We found that the amount of CR adsorbed to AG was significantly less than the amount adsorbed to β-1,3-glucan (BG) or chitin, suggesting that loss of cell-wall AG would increase exposure of BG on the cell surface, and thereby increase the sensitivity to CR. Generally, fungal BGs are known as biological response modifiers because of their recognition by Dectin-1 receptors in human immune systems. Therefore, isolation of AG-deficient mutants in Aspergillus oryzae has been used in the Japanese fermentation industry to create strains with increased ability to promote immune responses. Here, we aimed to isolate AG-deficient strains by mutagenizing A. oryzae conidia with chemical mutagens. Based on the increased sensitivity to CR in AG-deficient strains of A. nidulans and A. oryzae, we established a screening method for isolation of AG-deficient strains. Several candidate AG-deficient mutants of A. oryzae were isolated using the screening method; these strains showed increased sensitivity to CR and/or LE. Cytokine production was increased in the dendritic cells co-incubated with germinated conidia of the AG-deficient mutants. Furthermore, according to a Dectin-1 NFAT (nuclear factor of activator T cells)-GFP (green fluorescent protein) reporter assay, Dectin-1 response levels in the AG-deficient mutants were higher than those in wild-type A. oryzae. These results suggest that we successfully isolated AG-deficient mutants of A. oryzae with immunostimulatory effects.