Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms

Abstract

Edited by Gerald W. HartAspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear ␣-1,4 -linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative ␣-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting ␣-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (␤/␣) 8 -fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the ؊2 to ؉1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

“…8c) and are coregulated 5 . Recently, Ega3 was found to be specific for deacetylated regions of GAG supporting that agd3 and ega3 homologs are functionally linked 9 . Spherulin-4 annotated genes were also found in some of the Actinobacteria operons.…”

“…1) and includes the production of activated monosaccharide building blocks by the epimerase Uge3 5,7 followed by synthesis and export by the predicted integral membrane glycosyltransferase Gtb3 5 . The GAG cluster also encodes two glycoside hydrolases: an endoα-1,4-N-acetylgalactosaminidase, Sph3 6,8 ; and an endo-α-1,4galactosaminidase, Ega3 9 . Lastly, GAG is partially deacetylated by the secreted protein Agd3 and deletion of agd3 abolishes this modification 5 .…”

The mechanisms of hyphal pellet formation mediated by polysaccharides, α-1,3-glucan and galactosaminogalactan, in Aspergillus species

“…Bamford et al [98] recently reported that Ega3 has endo-α-1,4-galactosaminidase activity, but the function of Ega3 during GAG biosynthesis is still unknown. The authors suggested that the N-terminal transmembrane region of Ega3 is cleaved, and then the mature Ega3 form is released from the membrane and hydrolyzes deacetylated GAG [98] (Fig. 3b).…”

Unraveling the sugar code: the role of microbial extracellular glycans in plant–microbe interactions

“…As observed in the latter, the EPS architecture within such multipartite consortia is jointly shaped and highly adapted to the demands of its inhabitants. Taking into account that each partner within collaborative microbial biofilms contributes its own set of enzymes involved in glycan synthesis and hydrolysis ( Bamford et al ., 2019 ), this could fuel the generation of unique community patterns. Therefore, it would be of great interest to understand whether such community-derived glycan signatures are relevant for microbiota assembly and plant recognition.…”