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The conjugative relaxase TrwC delivers DNA into human cells and promotes its integration in the human genome

AutorGonzález-Prieto, Coral ; Gabriel, Richard; Schmidt, Manfred; Llosa, Matxalen
Fecha de publicación2016
CitaciónPlasmid Biology 2016
ResumenBacterial conjugation consists on the transfer of relaxase-DNA complexes between bacteria through a Type IV secretion system (T4SS). These systems stand out for their plasticity in terms of translocated substrates (proteins and/or DNA) and secretion target (prokaryotic or eukaryotic cells). We exploited this versatility to secrete a conjugative T4SS substrate through a T4SS required for effector translocation into human cells, resulting in efficient translocation of the relaxase-DNAinto human cells through the VirB/D4 T4SS of Bartonellahenselae (Fernández-González et al., 2011). This accomplishment suggests that natural transkingdom DNA transfer may occur through T4SS of human pathogens (Llosa et al., 2012). The DNA introduced in the human cell is led by TrwC, the conjugative relaxase of plasmid R388, responsible for DNA processing during bacterial conjugation. Apart from its role in conjugation, TrwC is also a site-specific integrase being able to catalyse integration of the mobilized DNA into a copy of its target sequence (oriT) present in the recipient cell (Agúndezet al., 2012). We have characterized integration of TrwC-driven DNA molecules into the recipient genome when a target oriT copy is not available, both in bacterial and in human genomes. We have selected stable integration events of the mobilized DNA into the human genome. TrwC was found to facilitate such integration by more than 3 logs. We have characterized the integration pattern mediated by TrwC using linear amplification-mediated PCR(LAM-PCR; Gabriel et al., 2014). We have identified at least one site-specific integration event mediated by TrwC, implyingTrwC is active in human cells; however, the vast majority of integrants represent random integration events of recircularized plasmid. Our results suggest that TrwC stabilizes the incoming DNA molecules, probably by protecting both ends, favoring their long-term presence and subsequent genomic integration through host-mediated mechanisms. For this reason, TrwC could be used in conjunction with a site-specific nuclease to accomplish targeted genomic modification of human cells.
DescripciónResumen del trabajo presentado al Plasmid Biology Meeting, celebrado en Cambridge (UK) del 18 al 23 de septiembre de 2016.
URIhttp://hdl.handle.net/10261/164776
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