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Bacteria synthesize hundreds of bacteria-specific or "rare" sugars that are absent in mammalian cells and enriched in 6-deoxy monosaccharides such as l-rhamnose (l-Rha). Across bacteria, l-Rha is incorporated into glycans by rhamnosyltransferases (RTs) that couple nucleotide sugar substrates (donors) to target biomolecules (acceptors). Since l-Rha is required for the biosynthesis of bacterial glycans involved in survival or host infection, RTs represent potential antibiotic or antivirulence targets. However, purified RTs and their unique bacterial sugar substrates have been difficult to obtain. Here, we use synthetic nucleotide rare sugar and glycolipid analogs to examine substrate recognition by three RTs that produce cell envelope components in diverse species, including a known pathogen. We find that bacterial RTs prefer pyrimidine nucleotide-linked 6-deoxysugars, not those containing a C6-hydroxyl, as donors. While glycolipid acceptors must contain a lipid, isoprenoid chain length, and stereochemistry can vary. Based on these observations, we demonstrate that a 6-deoxysugar transition state analog inhibits an RT in vitro and reduces levels of RT-dependent O-antigen polysaccharides in Gram-negative cells. As O-antigens are virulence factors, bacteria-specific sugar transferase inhibition represents a novel strategy to prevent bacterial infections.

Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.