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Infection and Immunity, March 2001, p. 1856-1868, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1856-1868.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Polarized Entry of Uropathogenic Afa/Dr Diffusely Adhering Escherichia coli Strain IH11128 into Human Epithelial Cells: Evidence for alpha 5beta 1 Integrin Recognition and Subsequent Internalization through a Pathway Involving Caveolae and Dynamic Unstable Microtubules

Julie Guignot,1,2 Marie-Françoise Bernet-Camard,1,2 Christian Poüs,2,3 Laure Plançon,4 Chantal Le Bouguenec,4 and Alain L. Servin1,2,*

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510,1 Laboratoire de Biochimie, EA1595,3 Institut Fédératif de Recherche, IFR75,2 Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, and Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris,4 France

Received 4 August 2000/Returned for modification 23 October 2000/Accepted 12 December 2000

Afa/Dr diffusely adhering Escherichia coli strain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the alpha 5beta 1 integrin and a pathway involving caveolae and dynamic microtubules (MTs). IH11128 invasion within HeLa cells was dramatically decreased after the cells were treated with the cholesterol-extracting drug methyl-beta -cyclodextrin or the caveola-disrupting drug filipin. Disassembly of the dynamically unstable MT network by the compound 201-F resulted in a total abolition of IH11128 entry. In apically infected polarized fully differentiated Caco-2/TC7 cells, no IH11128 entry was observed. The entry of bacteria into apically IH11128-infected fully differentiated Caco-2/TC7 cells was greatly enhanced by treating cells with Ca2+-free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated polarized Caco-2/TC7 cells grown on inverted inserts mounted in chamber culture showed a highly significant level of intracellular IH11128 bacteria compared with cells subjected to the apical route of infection. No expression of CD55 and CD66e, the receptors for the Afa/Dr adhesins, was found at the basolateral domains of these cells. Consistent with the hypothesis that a cell-to-cell adhesion molecule acts as a receptor for polarized IH11128 entry, an antibody blockade using anti-alpha 5beta 1 integrin polyclonal antibody completely abolished bacterial entry. Experiments conducted with the laboratory strain E. coli K-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, demonstrated that the dra operon is involved in polarized entry of IH11128 bacteria. Examined as a function of cell differentiation, the number of internalized bacteria decreased dramatically beyond cell confluency. Surviving intracellular IH11128 bacteria residing intracellularly had no effect on the functional differentiation of Caco-2/TC7 cells.

* Corresponding author. Mailing address: Unité 510 INSERM, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, France. Phone and fax: 33.1.46.83.56.61. E-mail: alain.servin{at}cep.u-psud.fr.

Infection and Immunity, March 2001, p. 1856-1868, Vol. 69, No. 3
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.3.1856-1868.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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