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

Le Mauff, F.; Bamford, N.C.; Alnabelseya, N.; Zhang, Y.; Baker, P.; Robinson, H.; Codee, J.D.C.; Howell, P.L.; Sheppard, D.C.

doi:doi:10.1074/jbc.RA119.008511

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 multi-functional 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 (Sph3_h and PelA_h, respectively) share structural and functional similarities given their ability to...Show moreDuring 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 multi-functional 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 (Sph3_h and PelA_h, 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 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 versus 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.Show less

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