Bacterial biofilms consist of surface attached bacterial cells embedded within an extracellular polymeric substance (EPS) composed of exopolysaccharides, proteins and extracellular DNA that facilitates cell–cell and cell–surface attachment and provides a protective barrier. Exopolysaccharides consisting of β-(1→6)-poly-N-acetylglucosamine(PNAG) is a critical component of the biofilm EPS in numerous gram-positive and gram-negative bacteria, including Staphylococcus aureus, Escherichia coli, and Acinetobacter baumanii. PNAG is partially deacetylated through the action of carbohydrate esterase enzymes during production. Degrading PNAG using specific glycosidase enzymes has emerged as a promising approach to disrupt existing biofilm. Understanding how these enzymes interact with their PNAG substrates is critical for their development as biocatalysts and will provide critical insight into the process of EPS breakdown during the natural process of biofilm dispersal. Specifically, it is not clear how PNAG deacetylation affects its recognition and degradation by glycosidase enzymes. In this study, we use synthetic acetylation defined PNAG substrate analogs to probe the mechanism of substrate recognition by Dispersin B (DspB), a PNAG specific exo-glycosidase from Actinobacillus actinomycetemcomitans. Synthetic PNAG trisaccharide analogs were prepared using an iterative one-pot glycosylation strategy using two protected glucosamine acceptors, followed by selective deprotection to obtain products with chemically defined acetylation patterns. Herein, we present the results of enzymatic breakdown of acetylation defined PNAG analogs by DspB and discuss the mechanistic implication of these results. These studies provide new insight into the specific recognition of deacetylated exopolysaccharides, and their degradation by DspB.

Probing the enzymatic breakdown of PNAG by DispersinB using acetylation defined substrates

2019 Fall National Meeting

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