UNIVERSITY OF WASHINGTON
This project is to determine how the intracellular metabolite cyclic diguanylate (c-di-GMP) stimulates bacterial biofilm formation. Our focus is on the opportunistic pathogen and model biofilm bacterium, Pseudomonas aeruginosa. Biofilms, defined as surface-associated multicellular communities encased in a self-produced exopolysaccharide (EPS) matrix, are responsible for chronic P. aeruginosa infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are problematic because they are resistant to antibiotic treatment and tend to escape immune surveillance. P. aeruginosa cells with high c-di-GMP form thick biofilms. Although the general parameters of c-di-GMP activity have been established, the mechanisms of c-di-GMP action are still unclear. A major effect of c-di-GMP in P. aeruginosa is to stimulate the activity of Pel EPS biosynthetic enzymes. We hypothesize that this due to the compartmentalized production of c-di-GMP at specific subcellular sites by specific diguanylate cyclases, one of which is WspR. WspR is a hybrid response regulator-GGDEF protein that functions in conjunction with proteins that encode a chemotaxis-like signal transduction complex. Fluorescent protein-tagged WspR forms dynamic clusters in the cytoplasm of cells when it is phosphorylated and therefore active. One of our three specific aims is to use fluorescence microscopy to analyze the subcellular distribution of WspR, its movement and its association with other proteins. In addition, fluorescence microscopy will be used to establish structural features of WspR itself that are important for its subcellular localization and in vivo function. WspR is a relatively low abundance protein that we are just barely able to detect with our current fluorescence microscope. This request is for an administrative supplement to upgrade our system with a new and more sensitive CCD camera and also accessories that will allow us to carry out fluorescence resonance energy transfer experiments (FRET) to visualize protein-protein interactions in vivo. There is now ample evidence that high c-di-GMP stimulates biofilm formation, a trait associated with chronic virulence, in P. aeruginosa. This suggests that the development of therapeutic agents that lower c-di-GMP levels in cells might be a promising avenue for preventing or treating chronic biofilm infections.