Polarized entry of uropathogenic Afa/Dr diffusely adhering Escherichia coli strain IH11128 into human epithelial cells: evidence for alpha5beta1 integrin recognition and subsequent internalization through a pathway involving caveolae and dynamic unstable microtubules.
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 alpha5beta1 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-alpha5beta1 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.
Guignot
J
J
Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510, France.
Bernet-Camard
M F
MF
Poüs
C
C
Plançon
L
L
Le Bouguenec
C
C
Servin
A L
AL
D018829:Adhesins, Bacterial / Q000235:genetics; D015703:Antigens, CD; D000943:Antigens, Differentiation; D001422:Bacterial Adhesion; D018958:CD55 Antigens; D021941:Caveolae; D015815:Cell Adhesion Molecules; D002454:Cell Differentiation; D016764:Cell Polarity; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology*; D004926:Escherichia coli / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000382:microbiology; D008870:Microtubules; D009876:Operon; D017469:Receptors, Fibronectin / Q000378:metabolism*; D014552:Urinary Tract Infections / Q000209:etiology
D018829:Adhesins, Bacterial; D015703:Antigens, CD; D000943:Antigens, Differentiation; D018958:CD55 Antigens; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D017469:Receptors, Fibronectin