Fate of plasmid DNA in host cells after bacteria-mediated gene transfer

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Bacteria as vehicles for the delivery of therapeutic DNA have several properties that the vectors commonly used to date cannot provide. Probably, the most import of such advantages is the independence of the vehicle and the expression plasmid. Thus, both can be manipulated independently. Gene delivery is achieved after intracellular lysis of the carrier bacteria. So far, the efficiency of DNA delivery is still rather low despite of the vast amount of plasmid DNA that is released into the host cell after bacterial lysis. Therefore, in the present project the fate of the expression plasmids after bacterial delivery by two bacteria - Listeria monocytogenes and Shigella flexneri - will be determined. First, the involvement of autophagic vesicles during bacterial infection and DNA release will be investigated. Autophagy represents a host defence system that is activated after invasion by bacteria and might seriously impair plasmid transfer. These experiments will be extended by cellbiological studies to follow the plasmids after release from the carrier. Especially the remaining association of the plasmids with bacterial proteins will be investigated. Finally, minimal expression cassettes or minimal episomal plasmids will be generated during the transfer by employing novel bacterial carriers with inducible recombinases. This should provide the knowledge for a rational design of highly efficient bacterial transfer vehicles.