Regulation of O‐antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O‐antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O‐antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)‐resistance cassette into the rol gene (controlling the modal O‐antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP‐rhamnose (the precursor of rhamnose in the O‐antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria‐infected HeLa cells stained with fluorescein isothiocyanate (FITC)‐phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F‐actin tail formation: the latter mutant showed distorted F‐actin tails. Plasma‐membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F‐actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell‐surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O‐antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O‐antigen chains, and comparing untreated and Sf6c‐treated cells by immunofluorescence with anti‐IcsA serum.