PROJECT SUMMARY/ABSTRACT: It is estimated that over 1.7 million hospital acquired infections per year in the U.S. result from microbial biofilms. New approaches to specifically target and disrupt bacterial biofilms will thus have a significant impact on human healthcare. Exopolysaccharides like poly-N-acetylglucosamine (PNAG) are critical biofilm components that facilitate cell-cell interactions and serve as a protective barrier against the host’s immune system and common antibiotic therapeutics used to treat bacterial infections. However, there is little is known about the molecular interactions of PNAG with other biofilm EPS components. Blocking the interactions between bacterial cells and PNAG or using glycosidase enzymes that break down PNAG and disrupt biofilms are attractive approaches to treat biofilm infections. Overall, our goal is to characterize molecular interaction networks of exopolysaccharides like PNAG, determine how they contribute to biofilm formation and dispersal, and develop novel strategies to treat biofilm infections by blocking these interaction networks or by catalyzing the breakdown of critical biofilm EPS components. To accomplish this overarching goal, we have identified two primary research areas for the next 5 years and plans for beyond. The first area seeks to develop tools for rapid identification of PNAG in bacterial biofilms and develop a live cell proximity labeling platform for identification and subsequent characterization of PNAG-binding proteins. These protein-carbohydrate binding interaction have the potential to be targets for new anti-biofilm therapeutics. A second area will develop high-throughput approaches to identify and subsequently engineer PNAG glycosidase enzymes through functional screening of environmental metagenomes and iterative site saturation mutagenesis approaches. To enable these studies, we have developed new colorimetric and fluorometric PNAG analogs that enable the high throughput identification of PNAG glycosidase activity. This research will establish a platform in my laboratory to rapidly identify and develop novel biocatalysts for biofilm dispersal activity. Overall, these research efforts will provide new unique insight into the role of PNAG in biofilm formation and will provide tools that will impact the way that we approach treating biofilm infections.