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. 2017 Aug 22;8:1503.
doi: 10.3389/fmicb.2017.01503. eCollection 2017.

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

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Free PMC article

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

Dolores L Guzmán-Herrador et al. Front Microbiol. .
Free PMC article

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.

Keywords: Bartonella henselae; Coxiella burnetii; Legionella pneumophila; bacterial conjugation; conjugative relaxase; gene therapy; intracellular pathogen; protein secretion.

Figures

FIGURE 1
FIGURE 1
Transient expression of transferred DNA in HeLa and EA.hy926 cells. Pools of cells obtained at 3 days post infection with B. henselae were analyzed by flow cytometry using uninfected cells as control. (A) Representative plots (cell granularity versus GFP intensity). The square marks the population considered as positive. The relaxase present in each experiment is indicated on top of the panels. (B) Mean values of the percentage of GFP positive cells from 2 to 4 independent experiments, after subtracting the background values of the negative controls with no relaxase. The relaxase present in each experiment is indicated.
FIGURE 2
FIGURE 2
DNA integration rate for different relaxases. HeLa cells were infected with B. henselae containing the plasmids encoding the indicated relaxases (or the negative controls lacking the relaxase) and at 3 days post infection, cells were either analyzed by flow cytometry (to determine transient expression of eGFP), or subjected to antibiotic selection (to determine permanent expression of the antibiotic resistance gene). The graph shows the ratio between the number of antibiotic-resistant cells and the number of GFP positive cells.
FIGURE 3
FIGURE 3
DNA transfer through the Dot/Icm T4BSS. (A) Representative flow cytometry plots for quantification of GFP positive CHO FcγRII cells after infection with L. pneumophila strains Lp02 (wild type, left panel) or Lp03 (dotA mutant, right panel) harboring plasmid pAA58, which encodes MobA. (B) Fluorescence microscope images showing GFP positive CHO FcγRII cells infected with L. pneumophila strains Lp02 (wild type, left panel) or CR503 (ΔicmSΔicmW mutant, right panel) harboring plasmid pAA12, which encodes TrwC-RalF. (C) Representative flow cytometry plots for quantification of GFP positive HeLa cells after infection with C. burnetii strains RSA439 intergenic::Tn (wild type, left panel) or RSA439 dotA::Tn (dotA mutant, right panel) harboring plasmid pAA58, which encodes MobA. ig, intergenic.
FIGURE 4
FIGURE 4
The presence of MobA interferes with intracellular replication of C. burnetii in HeLa cells. (A) Flow cytometry histogram analysis of HeLa cells infected with the indicated C. burnetii strains at 4 days post infection. The location of GFP positive cells (see Figure 3C and Materials and Methods) is indicated. (B) Quantification of intracellular replication of C. burnetii. Total genome equivalents (GE)/well are shown at two time points post infection for infections with the indicated strains at a MOI of 50. The bottom strain legend applies for (A,B). The presence or absence of plasmid-encoded MobA is indicated in brackets. ig, intergenic. hpi, hours post infection.
FIGURE 5
FIGURE 5
Possible fates of translocated DNA in a human cell. See text for details. Random DNA is represented by the jagged line. Mobile genetic element (MGE) is represented by the wavy line. RLX, relaxase (small yellow sphere). BT, beneficial trait (small orange box).

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