Project Summary Bacteria are most commonly found in nature in multicellular communities termed biofilms. Biofilms are formed when bacteria synthesize, secrete, and enmesh themselves with diverse biopolymers. Beneficial bacteria in the microbiome assemble biofilms, while biofilms are unfortunately also linked to difficult-to-treat infections that exhibit increased tolerance to antibacterials and can exhaust treatment options. However, there are no blueprints for how bacteria build these tissue-like architectures and uncovering these details can accelerate discovery of new anti-infectives. E. coli, in particular, are normal residents in the healthy microbiome, but emerge as pathogens when they egress and colonize the urinary tract. E. coli, Salmonella species and other Gram-negative organisms harness specific amyloid and polysaccharide machinery to elaborate mechanically robust extracellular matrix architectures resembling baskets and blankets that surround cells and drive the formation of tissue-like biofilms. Due to the complexity of biopolymer composites, there are significant challenges associated with studying their structure and function, yet the ubiquity of these biopolymers makes them of high importance for study. This research plan is directed to test molecular hypotheses for how bacteria employ curli and phosphoethanolamine cellulose, a newly discovered chemically modified form of cellulose, to enmesh themselves in extracellular matrix (ECM). The research plan will test hypotheses regarding functional roles that we propose are ascribed to the zwitterionic phosphoethanolamine modification. Aim 1 is directed to evaluate the temporal and spatial developments of matrix assembly beyond the bacterial cell surface using fluorescence microscopy and creative functional biochemical assays. Aim 2 will implement a strategically designed solid-state nuclear magnetic resonance (NMR) approach to detect molecular contacts between polysaccharides and protein amyloids that are responsible for matrix cohesion. The functional benefit of ECM biopolymers will be determined in Aim 3, where clinically relevant antibiotics and a novel vancomycin-conjugate will be evaluated for efficacy against pEtN cellulose and curli containing biofilms. This work promises to formulate a molecular foundation for future avenues of inquiry at the host-pathogen interface, involving possible immunomodulatory roles of bacterial polysaccharides and amyloids, and possible biopolymer contributions to microbiome symbiosis and amyloid- associated disease pathologies. The fellowship candidate will receive significant training in solid-state NMR spectroscopy to study molecular interactions within E. coli biofilms and biochemical approaches to investigate bacterial communities. The considerable support and mentorship structure provided through this fellowship, the research sponsor (Prof. Lynette Cegelski) and institution (Stanford University) will facilitate the professional development of t...