Detachment of Actinobacillus actinomycetemcomitans Biofilm Cells by an Endogenous β-Hexosaminidase Activity

Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus that colonizes the human oral cavity (20). A. actinomycetemcomitans has been implicated as the causative agent of localized juvenile periodontitis, a severe and rapid form of periodontal disease that affects adolescents (38). A. actinomycetemcomitans can also enter the submucosa and cause infective endocarditis and other nonoral infections (15). When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite (5, 6, 9, 12-14, 16, 18, 20, 28). Nearly all of the cells grow attached to the surface, while the broth remains clear and is often sterile (5). The dense biofilm that forms on the surface is resistant to removal by agents such as detergents, proteases, heat, sonication, and vortex agitation and can be removed only by mechanical scraping or by treatment with the carbohydrate-modifying reagent periodic acid (5). A. actinomycetemcomitans biofilm cells exhibit increased resistance to antimicrobial agents compared to the resistance exhibited by cells grown in planktonic form (4). Tight adherence has been shown to play an important role in the ability of A. actinomycetemcomitans to colonize the mouths of rats (7) and probably plays an equally important role in its ability to colonize humans. Tight adherence to surfaces is dependent on the presence of long, bundled pili (fimbriae) that form on the surface of the cell (12, 28). Mutations in flp-1, which encodes the major pilin protein subunit, result in cells that fail to produce fimbriae or adhere to surfaces (14). Kaplan and Fine showed that biofilm colonies of A. actinomycetemcomitans release cells into liquid medium and that these cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread (16). Kaplan et al. isolated three A. actinomycetemcomitans transposon insertion mutants that formed biofilm colonies which were tightly adherent to surfaces but which failed to release cells into the medium or spread over the surface (18). All three of the transposon insertions mapped to genes required for the synthesis of the O-polysaccharide (O-PS) component of lipopolysaccharide. Microscopic analysis of the O-PS mutants indicated that they lacked a layer of nonaggregated cells that was present inside biofilm colonies of the wild-type parental strain. These findings led to the hypothesis that A. actinomycetemcomitans biofilm cell detachment occurs by means of a novel mechanism that involves the release of cells from inside the biofilm colony (18). In this report we describe a fourth A. actinomycetemcomitans transposon insertion mutant that is deficient in biofilm cell detachment and biofilm dispersal. The transposon in this strain inserted into a novel gene, designated dspB, which encodes a protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. By using DspB protein purified from an overexpressing strain of Escherichia coli, we obtained evidence that dspB encodes a soluble N-acetylglucosaminidase that causes the detachment of cells from A. actinomycetemcomitans biofilm colonies.