8-azido-3,8-dideoxy-D-manno-octulosonic acid-mediated metabolic oligosaccharide engineering for specific labeling of diverse Enterobacterales

Current microbiome studies lack real-time or spatial information, often requiring large cohorts for longitudinal investigations. Real-time, in vivo molecular imaging would alleviate these shortcomings but is hampered by the limited labeling performance of current strategies. Here, we use the azido-modified sugar 8-Azido-3,8-dideoxy-D-manno-octulosonic acid (Kdo-N₃) to metabolically engineer the core oligosaccharide of Gram negative lipopolysaccharide, enabling conjugation with DBCO-fluorophores. We extend this approach by systematically screening multiple Enterobacteriaceae strains, optimizing labeling conditions, validating core-oligosaccharide-specific labeling using LPS knockout mutants, and demonstrating in vivo species-specific MOE-BCC labeling with Kdo-N₃ inside the murine gastrointestinal tract and subsequent in vivo imaging. Beyond Salmonella Typhimurium and Escherichia coli Nissle 1917, a broad range of Enterobacterales species were amenable to Kdo-N₃ sugar incorporation, potentially unlocking species-specific studies of Bacterial interaction in vitro and in vivo.