Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/11665
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Type: Journal article
Title: Regulation of O-antigen chain length is required for Shigella flexneri virulence
Author: Van Den Bosch, L.
Manning, P.
Morona, R.
Citation: Molecular Microbiology, 1997; 23(4):765-775
Publisher: WILEY
Issue Date: 1997
ISSN: 0950-382X
1365-2958
Statement of
Responsibility: 
Luisa Van Den Bosch, Paul A. Manning, Renato Morona
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 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.
Keywords: Hela Cells; Cell Membrane; Humans; Shigella flexneri; Actins; O Antigens; Bacterial Proteins; DNA-Binding Proteins; Transcription Factors; Mutagenesis, Insertional; Virulence; Genes, Bacterial
RMID: 0030004163
DOI: 10.1046/j.1365-2958.1997.2541625.x
Appears in Collections:Microbiology and Immunology publications

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