The two polymers behind one false identity, a new perspective on galactosaminogalactan and Aspergillus fumigatus biofilm formation

Author:

François Le Mauff 1,2, Joshua Kerkaert 3, Ira Lacdao 1,2, Marc Yacoub 4, Benjamin Wucher 5, Fabrice Gravelat 1,2, Pierre-Guy Millette 1,2,*, Mario Vergas 6, Lynne P. Howell 6,7, Carey Nadell 5, Robert Cramer 3, J. Stajich 4, Shizhu Zhang 8, Donald C. Sheppard 1,2.

Author address:

1 Departments of Microbiology and Immunology, Faculty of Medicine, McGill University, Montréal, Canada 2 Infectious Diseases in Global Health Program, Research Institute of the McGill University Health Centre, Montréal, Canada 3 Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 USA 4 Department of Microbiology and Plant Pathology, Institute for Integrative Genome Biology, University of California Riverside, Riverside, California, USA 5 Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA 8 Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China
* Current address: Faculty of Medicine, Université de Montréal, Montréal, Canada
Category: Fungal biology and genomics

Abstract:

Objectives: Biofilm formation in Aspergillus fumigatus has been reported to be dependent on the synthesis and secretion of galactosaminogalactan (GAG), a partially deacetylated heteropolymer of α-1,4-N-acetylgalactosamine (GalNAc) and galactose (Gal). GAG biosynthesis is believed to be mediated by the products of a 5-gene cluster located on chromosome 3 (cluster 3). However, the majority of enzymes in this cluster exhibit GalNAc-specific activity, suggesting that other enzymes might contribute to the synthesis and incorporation of Gal into GAG.
Methods: Blast searches identified a cluster of genes on chromosome 4 with similar functions to those found in cluster 3. Mutants deficient in the glycosyltransferases located within each cluster (gtb3, gtb4) were constructed. Biochemical characterization of GAG produced by each mutant was then carried out by gas chromatography coupled to mass spectrometry. Biofilm formation was assessed by crystal violet staining and confocal microscopy.
Results: Deletion of gtb4 resulted in a strain that formed fully adherent biofilms, but produced a GAG which was composed of a partially deacetylated homopolymer of GalNAc uniquely, a polyGalNAc. Deletion of gtb3 was associated with the inability to form an adherent biofilm, and a complete loss of GAG production. However, gene expression studies demonstrated that deletion of gtb3 was also associated with a complete repression of gtb4 expression. Therefore, to study the effects of gtb4 expression in isolation, gtb4 was constitutively expressed in a Δgtb3 background. Analysis of this strain revealed the production of a polymer of galactose and N-acetylgalactosamine which cannot be deacetylated by Agd3, the N-acetylgalactosaminogalactan. Biofilm microscopy demonstrated that polyGalNAc was largely adherent to the hyphal surface, while N-acetylgalactosaminogalactan was found within the biofilm intercellular matrix.
These findings suggest that A. fumigatus GAG is actually comprised of two separate polymers: a polymer of partially deacetylated GalNAc, produced by the gene products of cluster 3, which mediates adherent biofilm formation (partially deacetylated polyGalNAc), and a polymer composed largely of Gal with random insertion of low levels of GalNAc, produced by the gene products of cluster 4, which is dispensable for adhesion and largely found within the extracellular matrix (N-acetylgalactosaminogalactan).
Conclusion: This study highlights that GAG is actually comprised of two polymers with similar yet distinct compositions and functions. Further studies are required to determine the role that each of these two polymers plays in the multiple functions that have been described for GAG.

Abstract Number: 55

Conference Year: 2024

Conference abstracts, posters & presentations

Showing 10 posts of 17325 posts found.

  • Title

    Author

    Year

    Number

    Poster