Structural insights into PSL polysaccharide assembly and export to the cell surface via the Wzx/Wzy-dependent pathway.

Bacterial extracellular polysaccharides play a crucial role in mediating pathogen-host interactions and bacterial fitness via biofilm formation. The Wzx/Wzy-dependent pathway is the most prevalent and conserved strategy for polysaccharide biosynthesis. Psl (polysaccharide synthesis locus) is a key biofilm matrix polysaccharide in Pseudomonas aeruginosa PAO1, and its biosynthesis machinery is predicted to be a Wzx/Wzy-dependent biosynthesis system. Here, using Psl in PAO1 as a model strain, we determine the cryo-EM structures of the PslD-PslE complex. These structures reveal that PslD-PslE complex forms a continuous, protected conduit across the entire cell envelope. Further structural and functional analyses demonstrate that the polymerase PslJ, is likely localized in the membrane lumen formed by the octameric arrangement of PslE's transmembrane helical pairs. We propose a mechanistic model in which Und-PP-linked pentasaccharide units of Psl access PslJ through side portals in the PslE octamer, shielding the polymerization and translocation processes from degradation by PlsG, a periplasm-localized endoglycosidase. The iterative addition of incoming repeat units to the reducing terminus of the growing polysaccharide chain is hypothesized to drive Psl export through the channel, a mechanism that may be conserved across the Wzx/Wzy-dependent polysaccharide biosynthesis pathways.