Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation

Abstract

The mold Aspergillus fumigatus causes invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-d-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3 encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3 mutant in the presence of culture supernatants of the GAG-deficient Δuge3 mutant rescued the biofilm defect of the Δagd3 mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotina subphylum of the Ascomycota including a number of plant-pathogenic fungi and a single basidiomycete species, Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity

“…Our recent work on Aspergillus has identified a cluster of five genes that encode the proteins necessary for GAG biosynthesis (15). As with P. aeruginosa, we found that the product of one of these genes contains a glycoside hydrolase domain, Sph3 h , which is capable of hydrolyzing purified and cell wall-associated GAG (23).…”

“…For fungal burden analysis, lungs were homogenized in 5 mL of PBS containing protease inhibitor mixture (Roche), and aliquots were stored at −80°C until use. Pulmonary fungal burden was determined as described (15), and as detailed in SI Methods.…”

“…Biofilm formation provides a significant advantage to these organisms because the matrix mediates adherence to host cells (8,9) and aids in the resistance to both antimicrobial agents (10,11) and host-immune defenses (12,13). A. fumigatus biofilm formation depends on the cationic polysaccharide galactosaminogalactan (GAG), a heteroglycan composed of α1,4-linked galactose and N-acetyl-D-galactosamine (GalNAc) that is partially deacetylated (14,15). In comparison, P. aeruginosa has the genetic capacity to produce three biofilm exopolysaccharides: alginate, Psl and Pel (16).…”

Biofilm Exopolysaccharides of Pathogenic Fungi: Lessons from Bacteria

“…GAG is an ␣-1,4-linked heteropolysaccharide composed of galactose and partially deacetylated GalNAc that is absent in A. fumigatus spores but is produced in abundance by growing hyphae (8,11,14). GAG plays a critical role in the maintenance of the extracellular matrix of A. fumigatus biofilms (8).…”

“…GAG is a partially deacetylated heteropolymer of ␣-1,4-linked galactose and N-acetyl galactosamine, which mediates adhesion between both fungi and also to other surfaces as well as protection against host immune defenses (8 -11). GAG is synthesized by the protein products of a conserved cluster of five genes that is present in a number of plant and human fungal pathogens (11). Despite a lack of sequence homology with bacterial exopolysaccharide biosynthetic enzymes, there are striking similarities in the function and organization of these fungal exopolysaccharide biosynthetic proteins and the role of the resulting glycans in biofilm formation, drug resistance, and immune evasion.…”