The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

Abstract

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

“…These plasmids (pAA58 and pMTX808; Table 2 ) were introduced in B. henselae , and the resulting strains were used to infect both EA.hy926 and HeLa human cell lines. The former is derived from HUVEC cells, which are the natural target of B. henselae in vivo ; however, HeLa cells can also be infected by B. henselae with lower efficiency, and we showed that TrwC-mediated DNA transfer takes place to HeLa cells as well ( Gonzalez-Prieto et al, 2017 ). B. henselae carrying plasmids coding for either MobA or TrwC, or relaxase mutants as negative controls, were used for infections.…”

“…The conjugative relaxase TrwC can be translocated through the T4SS VirB/D4 of B. henselae to human cells, where it promotes the integration of the transferred DNA into the recipient genome ( Gonzalez-Prieto et al, 2017 ). In this work, we wanted to test whether this is a unique feature of TrwC and VirB/D4, or other systems can also be combined to deliver and integrate DNA into human cells.…”

“…TrwC can also catalyze the integration of the transferred DNA molecule into a target sequence present in the recipient bacterium ( Draper et al, 2005 ); moreover, the protein can catalyze integration into DNA sequences present in the human genome that resemble its natural target, the oriT ( Agundez et al, 2012 ), opening the possibility that this relaxase could work as a site-specific integrase in human cells ( Gonzalez-Prieto et al, 2013 ). Recently, we have shown that the relaxase TrwC is active in a human cell after delivery by the T4SS of Bartonella henselae , where it can promote the integration of foreign DNA into the human genome, although without site-specificity ( Gonzalez-Prieto et al, 2017 ). The integration rate of the foreign DNA introduced by TrwC was about 100 times higher compared to when it was introduced by the Mob relaxase from Bartonella cryptic plasmid pRGB1, or by transfection.…”

Bacterial injection machines: Evolutionary diverse but functionally convergent

“…The translocated DNA is covalently attached to the conjugative relaxase. They showed that certain relaxases promote random integration of the incoming DNA into the recipient human genome (Gonzalez‐Prieto, Gabriel, Dehio, Schmidt, & Llosa, ). Through protein engineering, relaxase chimeric proteins can acquire new functions, including the ability to integrate the attached DNA (Agundez et al, ).…”

“…into the recipient human genome(Gonzalez-Prieto, Gabriel, Dehio, Schmidt, & Llosa, 2017). Through protein engineering, relaxase chime-ric proteins can acquire new functions, including the ability to integrate the attached DNA (Agundez et al, 2018).…”