title: Regulation of O-antigen chain length is required for Shigella flexneri virulence.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, University of Adelaide, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Van den Bosch
authors.forename: P A
authors.initials: PA
authors.lastname: Manning
authors.forename: R
authors.initials: R
authors.lastname: Morona
mesh_terms: D000199:Actins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D019081:O Antigens / Q000737:chemistry*; D012763:Shigella flexneri / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D019081:O Antigens; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri