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

Abstract

During infection, the fungal pathogen <i>Aspergillus fumigatus</i> 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 <i>N</i>-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an <i>A. fumigatus</i> glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from <i>Pseudomonas aeruginosa</i> involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt <i>A. fumigatus</i> 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 (Sph3_h and PelA_h, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt <i>A. fumigatus</i> biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-<i>N-</i>acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelA_h was solved to 1.54 Å and structure alignment to Sph3_h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelA_h hydrolyzed partially deacetylated substrates better than Sph3_h, a finding that agrees well with PelA_h's highly electronegative binding cleft <i>versus</i> the neutral surface present in Sph3_h Our insight into PelA_h'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.