The characterization of mycobacterial transfection to eukaryotic cells
Quanquin, Natalie Michele
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Recently, much attention has been given to the use of bacteria as vectors for gene transfer into mammalian cells. Attenuated strains of Shigella flexneri, Listeria monocytogenes and Salmonella spp. persist only long enough to deliver a DNA plasmid to the host cell nucleus, with the subsequent expression of the encoded protein. This system is being explored as a means of DNA vaccination, as naked DNA vaccines are largely unsuccessful in humans. We have chosen to characterize the plasmid transfer ability of mycobacteria, an immunogenic bacterial species which targets and persists inside phagocytic monocytes, making it an attractive candidate for vaccine delivery. Using red (dsRed) and green fluorescent protein (gfp) expression as a means of visualizing and quantitating the efficiency of DNA transfer, we transformed the fast-growing Mycobacterium smegmatis with plasmids carrying the reporter genes under the control of either a mycobacterial or eukaryotic promoter. Upon the infection of both phagocytic and non-phagocytic cells lines, we were thus able to identify and distinguish expression by the bacteria and the host cell via microscopy and flow cytometry, and correlate gene transfer with different levels of infection. In an attempt to further elucidate the mechanism of DNA release from a mycobacterium to a eukaryotic cell, we discovered an increased level of plasmid transfer in cells infected with hyperconjugating M. smegmatis mutants. However, mutants displaying a decreased conjugation phenotype were still capable of transfecting cells, suggesting that other mechanisms must exist that allow the release of plasmids into the host cell. When these mutants were transformed with eukaryotic expression plasmids encoding influenza hemagglutinin, they were also shown to confer partial protection to mice from lethal challenge with the virus. To our knowledge, this represents the first demonstration of mycobacterial delivery of DNA to mammalian cells, and suggests that future studies should focus on a genetic approach of isolating mutants that enhance or eliminate plasmid transfer.
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