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

François Le Mauff,Natalie C Bamford,Noor Alnabelseya,Yongzhen Zhang,Perrin Baker,Howard Robinson,Jeroen D.C Codée,P Lynne Howell,Donald C Sheppard

doi:10.1074/jbc.ra119.008511

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.

Highlights

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses
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
We recently reported that the recombinant glycoside hydrolase domains from two microbial proteins, A. fumigatus Sph3 and Pseudomonas aeruginosa PelA, degrade GAG and disrupt A. fumigatus biofilms [19]

Summary

Introduction

The fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. Filamentous hyphae of A. fumigatus grow within biofilms: multicellular communities of organisms embedded in a self-produced extracellular matrix [4] This biofilm extracellular matrix plays several roles in the pathogenesis of invasive aspergillosis including mediating the adherence of hyphae to host tissues and enhancing resistance to antifungal drugs and host immune defenses [5,6,7,8]. GAG synthesis is thought to be Molecular mechanism of glycoside hydrolases Sph3h and PelAh initiated by the synthesis of UDP-GalNAc and UDP-Gal by the glucose-4-epimerase Uge3 [12]. These sugars are linked and exported through the action of the predicted glycosyltransferase Gtb, and the resulting polymer is partially deacetylated in the extracellular space by secreted Agd3 [13]. Consistent with these observations, GAG-deficient strains of A. fumigatus exhibit attenuated virulence in mouse models of invasive aspergillosis [17]

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