An Inactive Dispersin B Probe for Monitoring PNAG Production in Biofilm Formation

The bacterial exopolysaccharide poly-β-1,6-N-acetylglucosamine is a major extracellular matrix component in biofilms of both Gram-positive and Gram-negative organisms. We have leveraged the specificity of the biofilm-dispersing glycoside hydrolase Dispersin B (DspB) to generate a probe (Dispersin B PNAG probe, DiPP) for monitoring PNAG production and localization during biofilm formation. Mutation of the active site of Dispersin B gave DiPP, which was an effective probe despite its low affinity for PNAG oligosaccharides (KD ∼ 1–10 mM). Imaging of PNAG-dependent and -independent biofilms stained with a fluorescent-protein fusion of DiPP (GFP-DiPP) demonstrated the specificity of the probe for the structure of PNAG on both single-cell and biofilm levels, indicating a high local concentration of PNAG at the bacterial cell surface. Through quantitative bacterial cell binding assays and confocal microscopy analysis using GFP-DiPP, discrete areas of local high concentrations of PNAG were detected on the surface...