Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells

• Published in: Journal of controlled release Vol. 97; no. 2; pp. 371 - 381
• Main Authors: Fajac, I, Grosse, S, Collombet, J.-M, Thevenot, G, Goussard, S, Danel, C, Grillot-Courvalin, C
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 18.06.2004; Elsevier
• Subjects: Adhesins, Bacterial - biosynthesis; Adhesins, Bacterial - genetics; Bacterial Toxins - biosynthesis; Bacterial Toxins - genetics; Bacterial vectors; Biological and medical sciences; Bronchi - cytology; Cells, Cultured; Cystic fibrosis; Cystic Fibrosis - pathology; DNA delivery; Epithelial Cells - metabolism; Escherichia coli; Escherichia coli - genetics; Gene therapy; Gene Transfer Techniques; General pharmacology; Genetic Vectors; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Heat-Shock Proteins - biosynthesis; Heat-Shock Proteins - genetics; HeLa Cells; Hemolysin Proteins; Humans; Integrin beta1 - biosynthesis; Listeria monocytogenes; Medical sciences; Microscopy, Confocal; Microscopy, Electron; Mutation; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Transfection; Yersinia pseudotuberculosis; Bacterial vectors; Cystic fibrosis; DNA delivery; Gene therapy; Pharmaceutical technology; Respiratory disease; Escherichia coli; Metabolic diseases; Genetic disease; Infection; Gene; DNA; Bacteriosis; Digestive diseases; Bacteria; Epithelial cell; Vector; Enterobacteriaceae; Pancreatic disease
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/j.jconrel.2004.03.025

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 β1-integrins only on these cells and with the required interaction for cell entry of E. coli expressing the invasin with β1-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.