Structural and functional studies of glycosyl hydrolases governing Vibrio biofilm dispersal

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PROJECT SUMMARY/ABSTRACT Biofilms are surface-attached communities of bacteria surrounded by an extracellular protective matrix composed of polysaccharides, proteins, and nucleic acids. Biofilms protect bacterial pathogens from antibiotics and the host immune system as well as from predation, nutrient limitation, and physical insults while in environmental reservoirs. As important as the formation of biofilms, dispersal mechanisms allow bacteria to degrade the biofilm matrix and escape in response to changes in internal or environmental cues. Understanding how biofilms disperse is important in developing new strategies for combatting biofilm-related infections and antibiotic-resistant bacteria. The long-term goal of this research is to use a structure/function approach to understand the mechanisms of biofilm formation, adhesion, and dispersal at the molecular scale. The overall objective of this proposal is to understand the mechanism of biofilm dispersal using the model biofilm-forming bacterium Vibrio cholerae. Vibrio cholerae biofilms are composed of a secreted exopolysaccharide called Vibrio polysaccharide (VPS), along with secreted matrix proteins and extracellular DNA. The central hypothesis of this proposal is that RbmB, a secreted putative glycosyl hydrolase, is a key factor in the dispersal of Vc biofilms and that it digests VPS leading to breakdown of the extracellular matrix. We aim to understand the structure and mechanism of RbmB-associated VPS digestion by pursuing the following three specific aims: 1) Understand the VPS cleavage specificity, mechanism and kinetic properties of the putative glycosyl hydrolase RbmB from Vibrio cholerae; 2) Determine the three-dimensional structure and enzymatic mechanism of RbmB in cleaving VPS; and 3) Using in vitro and in vivo techniques, determine how RbmB activity leads to degradation of the biofilm and Vibrio cholerae dispersal. We will use a combination of enzymatic assays, site-directed mutagenesis, X-ray crystallography, and imaging of living Vibrio cholerae biofilms to complete these aims. Our rationale for the proposed work is that by understanding the structure and mechanism of RbmB, its specificity towards VPS, and the role of matrix proteins in biofilm dispersal, we will gain a basic understanding of how biofilms disperse. While this proposal focuses on Vibrio cholerae, we expect that these insights will be applicable to other bacterial pathogens who produce biofilms using secreted exopolysaccharides and matrix proteins. Results from this proposal will contribute to our understanding of glycosyl hydrolases aiding their potential therapeutic use in the treatment of antibiotic-resistant and pernicious bacterial infections. This work also promises new research possibilities in the development of specifically cleavable polysaccharide scaffolds for bioengineering and biomedical applications.