Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity

Abstract

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.

FIG. 1.

Characterization of A. actinomycetemcomitans biofilm dispersal mutant JK1023. (A to D) Colony morphology on agar (A and B) and colony morphology in broth (C and D) of wild-type strain CU1000 (A and C) and mutant strain JK1023 (B and D). Bars = 1 mm. (E) Adherence to polystyrene as measured by a 96-well microtiter plate adherence assay (14). The optical density at 590 nm [O.D. (590 nm)] is proportional to cell adherence. Control wells contained no cells. Strain JK1009 carries a transposon insertion in flp-1 that causes complete loss of surface attachment (14). The values are mean ± standard deviations for triplicate samples. (F) Detachment of cells from biofilm colonies grown attached to polystyrene rods. The optical density at 590 nm is proportional to cell detachment. Control wells contained no cells. The values are means ± standard deviations for six wells of each sample.

FIG. 2.

Comparison of residues 21 to 381 of the predicted amino acid sequence of DspB from A. actinomycetemcomitans strain CU1000 (top line) with the amino acid sequence of lacto-N-biosidase from L. lactis (GenBank accession no. AAK05592) (bottom line). The hyphens indicate gaps inserted to maximize the similarity between the sequences. The lines between the sequences indicate identical residues. The asterisks indicate amino acid residues discussed in the text.

FIG. 3.

Genetic complementation of the dspB mutation in A. actinomycetemcomitans strain JK1023: colony morphology on agar (A and B) and biofilm dispersal phenotype in broth (C and D) of strain JK1023 harboring vector plasmid pJAK16 (A and C) or the complementary plasmid pJK618 (B and D). Bar = 1 mm. The cells in panels C and D were grown in the wells of six-well microtiter plates (diameter, 35 mm; Falcon no. 353046) for 5 days and stained with crystal violet as previously described (16).

FIG. 4.

Purification and characterization of A. actinomycetemcomitans DspB protein expressed in E. coli. (A) Five micrograms of purified, thrombin-cleaved DspB was electrophoresed through an SDS-12% polyacrylamide gel and stained with Coomassie blue. The molecular masses (in kilodaltons) of protein standards electrophoresed in an adjacent lane are indicated on the left. (B) Glycosyl hydrolase activity of purified DspB with four 4-nitrophenyl-labeled synthetic substrates. The optical density at 405 nm [O.D. (405 nm)] is proportional to the amount of 4-nitrophenolate released in the reaction. The substrates tested were 4-nitrophenyl-N-acetyl-β-d-glucosaminide (graph 1), 4-nitrophenyl-N-acetyl-α-d-glucosaminide (graph 2), 4-nitrophenyl-N-acetyl-β-d-galactosaminide (graph 3), and 4-nitrophenyl-N-acetyl-α-d-galactosaminide (graph 4).

FIG. 5.

DspB-induced detachment and disaggregation of A. actinomycetemcomitans biofilm cells. (A) Detachment of cells from preformed biofilm colonies of mutant strain JK1023 grown attached to polystyrene rods. The rods were suspended for 24 h in broth media containing different concentrations of purified DspB. There was no detachment in the presence of heat-inactivated DspB (100°C, 3 min) (asterisk). The optical density at 590 nm [O.D. (590 nm)] was proportional to biofilm cell detachment. The values are means ± standard deviations for triplicate wells. (B) Detachment of preformed biofilm colonies of strain CU1000 grown in the wells of a 96-well microtiter plate. Biofilm colonies were treated for 6 h with different amounts of DspB, and the cells remaining attached to the surface were stained with crystal violet. The optical density at 590 nm was proportional to the amount of attached cells. The values are means ± standard deviations for triplicate wells. (C) Disaggregation of ethidium bromide-stained clusters of CU1000 cells by purified DspB. Stained clusters of cells were mock treated (top) or treated with 170 mg of purified DspB per ml (bottom) for 30 min, and then 50-μl portions of the treated cell clusters were pipetted onto a sheet of cellophane that was placed on a UV transilluminator and photographed through an orange filter. Clusters of cells appear as white spots on a dark background.