Abstract
We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.
Highlights
Bacterial Type IV secretion systems (T4SS) selectively deliver macromolecules to other cells or to the extracellular media
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)
We compared the ability of different relaxases to transfer DNA to mammalian cells and to promote DNA integration into the recipient genome when translocated by the same T4SS, VirB/D4
Summary
Bacterial Type IV secretion systems (T4SS) selectively deliver macromolecules to other cells or to the extracellular media. T4SS-Mediated Genetic Modification of Host Cells or eukaryotic cells This plasticity allows T4SS to be involved in bacterial processes as diverse as horizontal DNA transfer or virulence (Christie, 2016). The formers are homologous to the prototypical VirB T4SS of Agrobacterium tumefaciens and have been characterized extensively, both functionally and structurally (Chandran Darbari and Waksman, 2015). Members of this family form part of conjugative systems of plasmids such as R388 or RP4; others are encoded in the genomes of human pathogens such as Bartonella henselae (Bh), Brucella melitensis or Helicobacter pylori among others, and their main role is to inject virulence factors to the target human cell. Research on T4BSS structure and function lags behind T4ASS; extensive work has been done regarding the role of T4BSS-delivered effectors within human cell (Hubber and Roy, 2010; Rolando and Buchrieser, 2014; Personnic et al, 2016)
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