DNA transfer and genomic integration in human cells using Type IV Secretion Systems of human pathogens

Resumen del trabajo presentado al 7th Congress of European Microbiologists, celebrado en Valencia (España) del 9 al 13 de julio de 2017.[Backgrounds]: -Gene therapy strategies require methods to introduce DNA into specific human cell types and to integrate it in the genome for stable expression. -We have shown that any DNA can be introduced into specific human cell types as a protein-DNA complex through the Type IV Secretion System (T4SS) of Bartonella henselae by a mechanism resembling bacterial conjugation. [Objectives]: -To extend this DNA delivery system to T4SS from other human pathogens targeting different cell types. -To follow the fate of the delivered DNA and analyze the integration pattern. -To compare the efficiency of different conjugative systems. [Methods]: -Infection of human cultured cell lines with human pathogens containing mobilizable plasmids with markers for eukaryotic selection and different conjugative systems. -Evaluation of transient and permanent expression of introduced DNA by measuring GFP positive cells and neomycin resistant colonies, respectively. -Determination of the integration pattern of the foreign DNA in the human genome by LAM-PCR and high throughput sequencing. [Conclusions]: -We can deliver DNA into different human cell types through the T4SS of Bartonella henselae, Legionella pneumophila, and Coxiella burnetii, suggesting DNA delivery can be accomplished by many, if not all, bacterial T4SS. -The efficiency of transfer depends on the conjugative relaxase. -The relaxase TrwC promotes genomic integration of the incoming DNA.Peer Reviewe