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

Mol Microbiol. 1997 Feb;23(4):765-75. doi: 10.1046/j.1365-2958.1997.2541625.x.


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 tall 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Bacterial Proteins / metabolism
  • Cell Membrane / metabolism
  • DNA-Binding Proteins / metabolism
  • Genes, Bacterial
  • HeLa Cells
  • Humans
  • Mutagenesis, Insertional
  • O Antigens / chemistry*
  • Shigella flexneri / genetics
  • Shigella flexneri / pathogenicity*
  • Shigella flexneri / physiology*
  • Transcription Factors / metabolism
  • Virulence / genetics


  • Actins
  • Bacterial Proteins
  • DNA-Binding Proteins
  • O Antigens
  • Transcription Factors
  • virG protein, Shigella flexneri