Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases

Abstract

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h. Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

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

“…The structure of TM1410 was determined as part of a structural genomics effort and has not been functionally characterized. PelA h has recently been shown to be an endo-␣-1,4-N-acetylgalactosaminidase belonging to family GH166 (18,34,35). PelA is involved in the biosynthesis of the Pel polysaccharide, which is similar to the GAG polysaccharide in that it contains 1,4-linked GalNAc, and is partially deacetylated (36).…”

“…Previously, we showed that Sph3, like PelA h , is an endo-␣-1,4-N-acetyl-galactosaminidase that adopts a (␤/␣)-barrel fold (16,18). Both Sph3 and Ega3 are encoded within the GAG cluster.…”

“…A model of the GAG biosynthetic system mediated by the products of these genes has been proposed (6), and to date, three of the genes, uge3, agd3, and sph3, have been experimentally linked to GAG production and virulence (6,16,17). Uge3 is a bifunctional cytoplasmic uridine diphosphatase (UDP)-glucose-4-epimerase that mediates the production of UDP-GalNAc and UDP-Gal (7,17), Agd3 is a secreted protein required for the partial deacetylation of newly synthesized GAG polymer (6), and Sph3 is a GH135 member, with endo-␣-1,4-N-acetylgalactosaminidase activity, and is required for the production of GAG (16,18). The other two genes in the cluster, gtb3 and ega3, are predicted to encode an integral membrane glycosyltransferase and a second glycoside hydrolase (GH), respectively (6,16).…”

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

“…BCE_5588 is homologous to the UDP-glucose-4-epimerase, GalE, and a transposon screen published during the course of this study has already implicated this gene in biofilm formation [42]. As BCE_5582 was predicted by the Phyre 2 algorithm [30] to adopt a structure similar to the endo-α-1,4- N -acetylgalactosaminidase domain of P. aeruginosa PelA (PelA H Pa ; [43]), we refer to BCE_5582 as PelA H Bc herein. Since PelA encoded in the pelDEA DA FG operon is predicted to contain only a polysaccharide deacetylase domain (PelA DA ), and we found orthologs of pelA H -like genes within or adjacent to many of the pel operons (Fig.…”

“…Since the WFL binding assay demonstrated that the polymers produced by B. cereus and P. aeruginosa are comparable (Fig. 3), we used the endo-α-1,4- N -acetylgalactosaminidase PelA H Pa [43] in a reciprocal experiment to challenge pre-formed ATCC 10987 biofilms. This led to a concentration-dependent decrease in biofilm formation (EC 50 = 96.5 ± 1.1 nM; Fig.…”