The role of anionic amino acids in hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

Abstract

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity with de-N-acetylated PNAG oligosaccharides; however, there is still little known about the molecular interaction required for DspB to bind native de-N-acetylated PNAG polysaccharides. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the –1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

Superhydrophobic Nanocoatings as Intervention against Biofilm-Associated Bacterial Infections

“…PNAG also mediates intercellular adhesion, thereby promoting the accumulation of bacterial cells. Hence, PNAG is regarded as a major structural determinant of biofilms contributing to sustained, persistent and nosocomial bacterial infections [ 24 , 35 ].…”