Actinobacillus actinomycetemcomitans and several pathogenic bacteria attach to abiotic surfaces and produce exopolymers that immobilize the bacterial cells on these colonized surfaces. Such colonization of these cells leads to biofilms with metabolic and physiological capabilities distinct from individual cells. Notable unique property of these biofilms is their resistance to antimicrobials. To colonize other virgin surfaces and to overcome starvation associated with overpopulation, cells must detach and disperse from these surfaces. We have previously shown that in Aa, the enzyme dispersin B is capable of assisting in the dispersal process. Additional studies by our group have shown that dispersin B is capable of preventing surface attachment of several related and non-related bacterial species. Since several pathogenic infections are caused by biofilms, new approaches in controlling the biofilm formation that focus on initial attachment of pathogenic bacteria on to the surfaces, in lieu of their antimicrobial resistance, are necessary. In this regard, study of enzymes that prevent the attachment of bacteria to surfaces through the use of extracellular polymeric substances is an underdeveloped area. We show that dispersin B depolymerizes exopolysaccharides that are made up of beta-1,6-linked N-acetylglucosamine (NAG), and is a potential candidate to remove biofilms of many Gram negative and Gram positive bacterial pathogens. Therefore, our long-range goals are to lay the foundation in the development of dispersin B as a broad spectrum anti-biofilm agent. Our immediate focus is on the structure-function studies on this enzyme to elucidate structural determinants that are necessary for its hydrolytic activity. We propose to achieve this through an integrated approach using x-ray crystallographic and mutational analysis.
The Specific Aims are: 1. Identify structural determinants governing the mechanism of action of dispersin B. 2. Map the substrate binding site through kinetic analysis using oligomeric substrate of beta-1,6-linked NAG. 3. Define the roles of specific amino acid residues in the enzymatic activity of dispersin B.
