title: 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.
  
  
  OBJECTIVE
  This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Lee
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Geller
authors.affiliation: Program in Molecular Structure & Function, The Hospital for Sick Children, Toronto, Canada Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Canada.
authors.forename: Natalie C
authors.initials: NC
authors.lastname: Bamford
authors.affiliation: Division of Infectious Diseases, Los Angeles Biomedical Medical Institute at Harbor-University of California, Los Angeles Medical Center, Torrance, California, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Fabrice N
authors.initials: FN
authors.lastname: Gravelat
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Brendan D
authors.initials: BD
authors.lastname: Snarr
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Le Mauff
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Joseé
authors.initials: J
authors.lastname: Chabot
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ralph
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Hanna
authors.initials: H
authors.lastname: Ostapska
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Lehoux
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Cerone
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada.
authors.forename: Stephanie D
authors.initials: SD
authors.lastname: Baptista
authors.affiliation: National Research Council, Ottawa, Canada.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Vinogradov
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside, California, USA.
authors.forename: Jason E
authors.identifier: http://orcid.org/0000-0002-7591-0020
authors.initials: JE
authors.lastname: Stajich
authors.affiliation: Division of Infectious Diseases, Los Angeles Biomedical Medical Institute at Harbor-University of California, Los Angeles Medical Center, Torrance, California, USA.
authors.forename: Scott G
authors.initials: SG
authors.lastname: Filler
authors.affiliation: Program in Molecular Structure & Function, The Hospital for Sick Children, Toronto, Canada Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Canada.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
authors.affiliation: Departments of Medicine and of Microbiology and Immunology, McGill University, Montréal, Canada Infectious Diseases and Immunity in Global Health Program, Research Institute of the McGill University Health Centre, Montreal, Canada don.sheppard@mcgill.ca.
authors.forename: Donald C
authors.initials: DC
authors.lastname: Sheppard
mesh_terms: D000107:Acetylation; D001228:Aspergillosis / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D018441:Biofilms; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; C062157:galactosaminogalactan