A dedicated C-6 β-hydroxyacyltransferase required for biosynthesis of the glycolipid anchor for Vi antigen capsule in typhoidal Salmonella

Vi antigen is an extracellular polysaccharide produced by Salmonella enterica Typhi, Citrobacter freundii, and some soil bacteria belonging to the Burkholderiales. In Salmonella Typhi, Vi-antigen capsule protects the bacterium against host defenses, and the glycan is used in a current glycoconjugate vaccine to protect against typhoid. Vi antigen is a glycolipid assembled in the cytoplasm and translocated to the cell surface by an export complex driven by an ABC transporter. In Salmonella Typhi, efficient export and cell-surface retention of the capsule layer depend on a reducing terminal acylated-HexNAc moiety. Although the precise structure and biosynthesis of the acylated terminus has not been resolved, it distinguishes Vi antigen from other known glycolipid substrates for bacterial ABC transporters. The genetic locus for Vi antigen-biosynthesis encodes a single acyltransferase candidate (VexE), which is implicated in the acylation process. Here, we determined the structure of the VexE in vitro reaction product by mass spectrometry and NMR spectroscopy to reveal that VexE catalyzes β-hydroxyacyl-ACP dependent acylation of the activated sugar precursor, uridine-5′-diphospho-GlcNAc, at C-6 to form UDP-6-O-[β-hydroxymyristoyl]-α-d-GlcNAc. VexE belongs to the lysophosphatidyl acyltransferase family, and comparison of an Alphafold VexE model to solved lysophosphatidyl acyltransferase structures, together with modeling enzyme:substrate complexes, led us to predict an enzyme mechanism. This study provides new insight into Vi terminal structure, offers a new model substrate to investigate the mechanism of glycolipid ABC transporters, and adds biochemical understanding for a novel reaction used in the synthesis of an important bacterial virulence factor.