Paper: An Analysis of the Functional Gene Transfer From Intracellular Bacteria

Functional gene transfer from intracellular bacteria to mammalian cells. Catherine Grillot-Courvalin, Sylvie Goussard, Francois Huetz, David M. Ojcius, and Patrice Courvalin. In the September issue of Nature Biotechnology in 1998, the above authors presented evidence of the first successful conjugative plasmid transfer from bacteria to mammalian cells. The authors have shown that E. coli, which underwent lysis upon entry into mammalian cells, can release plasmid DNA upon infection of the mammalian cells. This was made possible by conferring on a strain of the extracellular E. coli, which had a deficiency in cell wall biosynthesis, the gene for intracellular mobility taken from the Yersinia pseudotuberculosis which is able to transfer genes in mammalian cells. These recombinant E. coli may provide a new vector for DNA vaccine delivery. The results gave evidence that the invasive E. coli can directly transfer genetic material at a high level of efficiency into a variety of mammalian cells provided that they are able to penetrate the host mammalian cell. The authors hypothesis that two steps were required for bacteria to act as DNA delivery systems into mammalian cells: entry into the cell, followed by escape of the bacteria or of their DNA from the vacuole to the cytosol was proved accurate. Transfer of plasmid DNA was detected in 5-20% of the cells 2 days after invasion but did not always correlate with the initial number of internalized bacteria. A higher bacteria-to-cell ratio enhanced transfer efficiency, and it was found that transfer efficiency in some cell lines was improved with the co-production of the thiol-activated listeriolysin O from Listeria monocytogenes, which is largely responsible for the escape of Listeria from the entry vesicle. Listeriolysin is active only a low pH, as in phagolysosomes. Therefore these observations are consistent with the above hypothesis that the bacteria are lysed within the phagolysosomes, resulting in release of the listeriolysin which then triggers pore formation in the vacuolar membrane and subsequent release of DNA, which could then gain access to the cell nucleus. The experiment was preformed by growing E. coli strains at 30C in brain heart infusion broth then co- incubating them with mammalian cells for 2 days. Also incubated with them was a plasmid that directs synthesis of the green fluorescent protein (GFP) in mammalian cells, but not bacteria. Then the cells were analysed by fluorescence-activated cell sorting (FACS) and the percentage of cells expressing GFP varied form 4.9-20.4%. Flourescently labeled samples were examined with a Leica confocal microscope attached to a Leitz dioplan microscope equipped with a double argon-krypton laser. ...