Structural and functional studies of glycosyl hydrolases governing Vibrio biofilm dispersal - 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.
