The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread

Authors

  • Michelle Rathman,

    Corresponding author
    1. Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
    • *For correspondence. E-mail mrathman@ pasteur.fr; Tel. (+33) 1 40 61 32 47; Fax (+33) 1 45 68 89 53.

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  • Noureddine Jouirhi,

    1. Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, Route de Lennik 808, CP614, 1070 Bruxelles, Belgium.
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  • Abdelmounaaïm Allaoui,

    1. Laboratoire de Bactériologie Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, Route de Lennik 808, CP614, 1070 Bruxelles, Belgium.
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  • Philippe Sansonetti,

    1. Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
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  • Claude Parsot,

    1. Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
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  • Guy Tran Van Nhieu

    1. Unité de Pathogénie Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
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Abstract

In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cells. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)-based technology, we developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect the structure of their lipopolysaccharide or the ability of the bacteria to move intracellularly via actin-based motility; both factors have previously been shown to be essential for cell-to-cell spread. However, using a modified strategy that eliminated both of these types of mutants, we identified several mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into host cells, also play a role in cell-to-cell spread.

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