Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.

Keywords: Aspergillus; Sph3; biofilm; carbohydrate biosynthesis; crystal structure; galactosaminogalactan; glycoside hydrolase; in vivo imaging; mutagenesis in vitro; polysaccharide.

FIGURE 1.

Proposed GAG biosynthetic system and current model of the Ica system of Staphylococcus epidermidis. A, diagram of the co-regulated five gene cluster located on chromosome 3 involved in GAG biosynthesis. B, schematic representation of the ica operon from S. epidermidis. C, comparative model of the biosynthetic pathways of GAG and PNAG/polysaccharide intercellular adhesin polysaccharides from A. fumigatus and S. epidermidis, respectively. M, membrane; CW, cell wall; ECM, extracellular matrix. The synthase complex is red; deacetylase is green; epimerase is teal, and other modifying enzymes are tan.

FIGURE 2.

Sph3 contains a spherulin 4 family domain. A, schematic model of the Sph3 protein based on bioinformatics shows an N-terminal transmembrane domain and a C-terminal spherulin 4 domain. B, primary sequence alignment using ClustalW of spherulin 4 proteins from A. fumigatus, A. clavatus, Marssonina brunnea (Uniprot K1WTQ0), Ralstonia pickettii (Uniprot A0A080W3T6), and P. polycephalum (Lav6-1). Sequence identity as compared with Sph3_Af is listed. Conservation of amino acids is shown using gray scale with darker columns indicating higher conservation.

FIGURE 3.

Sph3 is required the production of secreted and cell wall-associated GAG. A, germination rate of the Δsph3 mutant as compared with wild-type A. fumigatus as determined by serial microscopy. B, radial growth rate of the Δsph3 mutant and wild-type A. fumigatus. C, cell wall-associated GAG production by the indicated strains was detected by SBA lectin staining (green). Hyphae were counterstained with DRAQ5 (red) for visualization in confocal images. D, scanning electron microscopy images of hyphae of each indicated strain. Wild-type A. fumigatus produces surface decorations associated with GAG production, whereas the Δsph3 mutant and GAG-deficient Δuge3 mutant lack these structures. E, quantification of secreted GAG in culture supernatants of the indicated strains using an indirect EIA.

FIGURE 4.

Structure of Sph3_Ac(54–304) and investigation of conserved regions. A, Sph3_Ac shown in schematic representation with transparent surface model reveals a (β/α)₈ barrel fold. The surface β-strands (purple) and α-helices (blue) are labeled β1–8 and α1–8, respectively. Loop regions are shown in light gray, and the ethylene glycol molecules are depicted as orange sticks. B, representation of the conserved surface as calculated by the ConSurf server (49–51) reveals a conserved groove on the top-face (C termini of β-strands) of the (β/α)₈ barrel with a small depression in the center. Conserved residues are shown in fuchsia and variable residues in teal. C, transparent surface representation of the Sph3_Ac structure (teal) in complex with GalNAc (yellow). D, close-up of the |F_o − F_o| omit density map contoured around GalNAc at σ = 2.5. E, superposition of the (β/α)₈ barrels of Sph3 (purple) and a representative member of GH18 (blue, PDB 4WIW), GH27 (yellow, PDB 1KTB), and GH84 (green, PDB 2CBJ) shows the similarity in the overall folds. Right panel, close-up of the active sites of these glycoside hydrolases reveals three acidic residues of Sph3 (Asp-166, Glu-167, and Glu-222) in the same vicinity. E, comparison of the ethylene glycol molecule (orange) found in the active site of the apo-structure (purple) with GalNAc (yellow) from the co-crystal structure (teal). Residues connected with black lines represent those within hydrogen bonding distance.

FIGURE 5.

Sph3 hydrolyzes purified and cell wall-associated GAG. A, Sph3 hydrolysis of purified GAG as measured by the release of reducing sugars. Purified GAG was incubated with 12 μm of the indicated proteins for 24 h. Activity is shown as difference between untreated and treated samples. *, p < 0.05 as compared with untreated levels with n = 3. B, treatment of chitosan with 10 μm Sph3_Ac for 24 h yielded no increase in reducing ends as compared with untreated polysaccharide. C, degradation of cell wall-associated GAG by the indicated Sph3 protein variants. A. fumigatus hyphae were treated with 0.05 μm of the indicated recombinant Sph3 variant for 3 h and then residual GAG was detected by SBA lectin staining (green). Bars represent means, and error bars are one S.D.

FIGURE 6.

Sph3 activity is required for functional GAG production. A, levels of Sph3 mRNA were measured using RT quantitative PCR for each indicated strain. B, SBA (green) staining for GAG and DRAQ5 (red) for DNA on indicated fungal strains. Af293 and the Δsph3 images as seen in Fig. 3A have been reproduced here for ease of comparison. C, hyphal morphology shown using S.E. images of indicated fungal strains. For comparison, the S.E. images of Af293 and Δsph3 from Fig. 3B have been included. D, absorbance readings of an indirect EIA using the anti-GAG antibody indicate cell filtrate GAG levels. Bars represent means, and error bars are one S.D.