Depolymerization of beta-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms

Abstract

Polymeric beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc) has been implicated as an Escherichia coli and Staphylococcus epidermidis biofilm adhesin, the formation of which requires the pgaABCD and icaABCD loci, respectively. Enzymatic hydrolysis of poly-beta-1,6-GlcNAc, demonstrated for the first time by chromatography and mass spectrometry, disrupts biofilm formation by these species and by Yersinia pestis and Pseudomonas fluorescens, which possess pgaABCD homologues.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of DspB and TLC separation of products generated by treatment of PGA with DspB. (A) Protein molecular weight standards (Stds.) and 10 μg of recombinant DspB were separated on a 12% polyacrylamide gel and stained with Coomassie blue R-250. (B) Reaction products generated by digestion of PGA for 16 h at 37°C are shown, along with untreated PGA and DspB controls. The GlcNAc standards are β-1,4-linked oligosaccharides derived from chitin.

FIG. 2.

MALDI-TOF mass spectra of PGA after digestion with DspB for 16 h at 37°C (A) and untreated PGA and DspB (B). The degree of polymerization of a major reaction product is indicated by a boxed number that refers to either the centroid mass (m/z) or ion peak of the spectrum.

FIG. 3.

Effect of DspB on biofilm formation by the following strains with the listed growth conditions: E. coli MG1655, LB medium, 26°C (A); TRMG1655 (csrA::kan), LB medium, 26°C (B); E. coli P18, TSB, 26°C (C); S. epidermidis 1457, TSB, 26°C (D); P. fluorescens WCS365, M63 supplement, 26°C (E); Y. pestis KIM6+, TSB, 26°C (F); S. enterica serovar Typhimurium, colony-forming antigen medium, 26°C (G); and P. aeruginosa PAO1, YPG, 26°C (H). Biofilm formation in the indicated media, in presence or absence of recombinant DspB or an enzyme buffer control, was determined at the indicated times by crystal violet staining (A₆₃₀). Error bars represent standard deviations (six replicates) of a representative experiment.

FIG. 4.

Treatment of biofilms of E. coli MG1655 (A), TRMG1655 (csrA::kan) (B), E. coli P18 (C), S. epidermidis 1457 (D), P. fluorescens WCS365 (E), S. enterica serovar Typhimurium (F), and P. aeruginosa PAO1 (G) with DspB or a buffer control (M63 salts). Biofilms were grown under the conditions indicated in the legend to Fig. 3. Error bars represent standard deviations (six replicates) of a representative experiment.