Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells.
To transfer genes into airway epithelial cells, we have generated auxotrophic dap Escherichia coli BM2710 mutant that expresses the invasin of Yersinia pseudotuberculosis and the listeriolysin of Listeria monocytogenes. E. coli BM2710 harboring a plasmid carrying the gfp gene was incubated with immortalized normal or cystic fibrosis (CF) airway epithelial cells or with primary bronchial epithelial cells grown as an explant-outgrowth cell culture model. Approximately 2% of immortalized cells expressed GFP. Few primary cells were transfected that were always poorly differentiated and located at the edge of the outgrowth. This was consistent with the expression of beta1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with beta1-integrins. The subsequent intracellular trafficking of E. coli BM2710 studied by confocal and electronic microscopy showed that the E. coli-containing phagosomes rapidly matured into phagolysosomes. This is the first demonstration that recombinant bacteria are able to transfer genes into primary airway epithelial cells, provided that they are able to invade the cells.
Fajac
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I
Laboratoire de Physiologie Respiratoire, Faculté de Medecine Cochin, AP-HP-Université Paris 5, IFR Alfred Jost, CHU Cochin, 24 rue du fg St. Jacques, 75014 Paris, France. ifajac@cochin.univ-paris5.fr
Grosse
S
S
Collombet
J-M
JM
Thevenot
G
G
Goussard
S
S
Danel
C
C
Grillot-Courvalin
C
C
D018829:Adhesins, Bacterial / Q000096:biosynthesis / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D001980:Bronchi / Q000166:cytology*; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006460:Hemolysin Proteins; D006801:Humans; D019012:Integrin beta1 / Q000096:biosynthesis; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009154:Mutation; D014162:Transfection
D018829:Adhesins, Bacterial; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D019012:Integrin beta1; C485184:enhanced green fluorescent protein; C053476:invasin, Yersinia; D049452:Green Fluorescent Proteins; C038705:hlyA protein, Listeria monocytogenes