Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms

Abstract

We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

Fig. 1.

Immunological detection of cell-bound PNAG. (a) Crude exopolysaccharide extracted from Burkholderia spp. (b) Effect of dispersin B (25 μg) on PNAG from crude extracts of B. cenocepacia and B. multivorans. Crude exopolysaccharide extracted from E. coli TRMG1655 was used a as positive control. (c) Effect of complementation of E. coli TRXWMGΔC(ΔpgaC) with pMCSpgaBCD on PNAG in exopolysaccharide extract.

Fig. 2.

Comparison of the biofilm dispersal effect of dispersin B on wild-type B. multivorans with that on the pgaBC mutant. Values are means ± standard deviations from two experiments with six replicates per sample. *, P < 0.01 compared with untreated control biofilm.

Fig. 3.

Effect of untreated control (▪) and dispersin B (□) on biofilm formation (a) and biofilm dispersal (b) in B. multivorans (Bm), B. vietnamiensis (Bv), B. cepacia (Bc), and B. cenocepacia (Bcn). The values are means ± standard deviations. *, P < 0.01 compared with untreated control biofilm.

Fig. 4.

Confocal image of B. multivorans biofilm formation on plastic coverslip. (a) Wild-type biofilm in the absence of dispersin B; (b) wild-type biofilm in the presence of 200 μg/ml dispersin B; (c) B. multivorans Mu5 (ΔpgaBC) biofilm in the absence of dispersin B. The images are maximum projections or reconstructed confocal stacks consisting of a series of xy (center), yz (left), and xz (bottom) sections. A representative CSLM image for each sample is shown.