Termination of Poly- <i>N</i> -acetylglucosamine (PNAG) Polymerization with <i>N</i> -Acetylglucosamine Analogues

Abstract

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In <i>E. coli</i>, the glycosyltransferase complex PgaCD produces poly-<i>N</i>-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N₃, SH, NH₂) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. <i>In vitro</i>, the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. <i>In vivo</i>, expression of the GlcNAc-1-kinase NahK in <i>E. coli</i> provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors <i>in situ</i>. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary <i>in vivo</i> mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.