PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms

Abstract

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

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

“…Recently, a similar requirement for a deacetylated moiety to help orient the substrate was found for the endo-acting glycoside hydrolase PgaB, which hydrolyzes partially deacetylated PNAG polysaccharide. PgaB requires the sequence of GlcN-GlcNAc-GlcNAc in the Ϫ3 to Ϫ1 subsites for substrate binding and cleavage (52).…”

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries

“…Deacetylation of PNAG is carried out by pgaB and icaB loci and has been shown to play a crucial role in biofilm formation and immune evasion(52,58). pgaB also possesses an additional C-terminal glycoside hydrolase domain which cleaves the PNAG polymer following its partial deacetylation(59), although the mechanism of how these activities are coordinated and the biological role of the hydrolase activity is unknown. Unique to pga operons is a loci encoding an outer membrane export pore, pgaA(60) , and in ica operons an additional integral membrane protein, icaC , which has been proposed to be involved in PNAG O-succinylation(53).…”

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries

“…Deacetylation of PNAG is carried out by pgaB and icaB loci and has been shown to play a crucial role in biofilm formation and immune evasion [51,70]. pgaB also possesses an additional C-terminal glycoside hydrolase domain which cleaves the PNAG polymer following its partial deacetylation [71], although the mechanism of how these activities are coordinated and the biological role of the hydrolase activity is unknown. Unique to pga operons is a locus encoding an outer membrane export pore, pgaA [72] and, in ica operons, an additional integral membrane protein, icaC, which has been proposed to be involved in PNAG O-succinylation [67].…”