<i>Agrobacterium</i>-Mediated T-DNA Transfer and Integration by Minimal VirD2 Consisting of the Relaxase Domain and a Type IV Secretion System Translocation Signal

Kregten, Maartje van; Lindhout, Beatrice I.; Hooykaas, Paul J. J.; Zaal, Bert J. van der

doi:10.1094/mpmi-22-11-1356

Cited by 47 publications

(49 citation statements)

References 45 publications

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“…These results support a correlation between Mob transfer rates and gene transfer rates and confirm the role of the relaxase as a pilot protein for the translocation of the plasmid DNA via the VirB/VirD4 T4SS (Fig. 1E), similarly as already demonstrated for the VirD2 protein during the delivery of the VirD2/T-DNA complex into plant cells (22).…”

Section: B Henselaesupporting

confidence: 87%

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Schröder

Schuelein²,

Québatte³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial type IV secretion systems (T4SS) mediate interbacterial conjugative DNA transfer and transkingdom protein transfer into eukaryotic host cells in bacterial pathogenesis. The sole bacterium known to naturally transfer DNA into eukaryotic host cells via a T4SS is the plant pathogen Agrobacterium tumefaciens . Here we demonstrate T4SS-mediated DNA transfer from a human bacterial pathogen into human cells. We show that the zoonotic pathogen Bartonella henselae can transfer a cryptic plasmid occurring in the bartonellae into the human endothelial cell line EA.hy926 via its T4SS VirB/VirD4. DNA transfer into EA.hy926 cells was demonstrated by using a reporter derivative of this Bartonella -specific mobilizable plasmid generated by insertion of a eukaryotic e gfp -expression cassette. Fusion of the C-terminal secretion signal of the endogenous VirB/VirD4 protein substrate BepD with the plasmid-encoded DNA-transport protein Mob resulted in a 100-fold increased DNA transfer rate. Expression of the delivered e gfp gene in EA.hy926 cells required cell division, suggesting that nuclear envelope breakdown may facilitate passive entry of the transferred ssDNA into the nucleus as prerequisite for complementary strand synthesis and transcription of the e gfp gene. Addition of an eukaryotic neomycin phosphotransferase expression cassette to the reporter plasmid facilitated selection of stable transgenic EA.hy926 cell lines that display chromosomal integration of the transferred plasmid DNA. Our data suggest that T4SS-dependent DNA transfer into host cells may occur naturally during human infection with Bartonella and that these chronically infecting pathogens have potential for the engineering of in vivo gene-delivery vectors with applications in DNA vaccination and therapeutic gene therapy.

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Section: B Henselaesupporting

confidence: 87%

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Schröder

Schuelein²,

Québatte³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…These finding suggest that Ats-1's C terminus suffices to mediate transfer of the Cre reporter but that additional internal motifs might contribute to optimal transfer. Synergistic contributions of C-terminal TSs and internal motifs have been reported for other T4SS effectors (70)(71)(72).…”

Section: ϫ8mentioning

confidence: 60%

“…Mutational analyses of the AADs of E. coli TraD F and TrwB R388 (18,89) and, more recently, A. tumefaciens VirD4 At and Enterococcus faecalis PcfC pCF10 (19) provided genetic evidence for their contributions to substrate discrimination. The AADs of VirD4 At and PcfC pCF10 also bind the VirD2 At and PcfG pCF10 relaxases (19) that are responsible for processing and then piloting DNA cargoes through the A. tumefaciens VirB/ VirD4 and E. faecalis Prg/Pcf T4SSs, respectively (70,90). VirD2 carries a charged C terminus, the deletion of which abolishes translocation of VirD2 and T-DNA substrates to plant cells (70).…”

Section: Discussionmentioning

confidence: 99%

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Whitaker¹,

Berry²,

Rosenthal³

et al. 2016

J Bacteriol

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Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4 At ) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4 At and the TraJ/VirD4 At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ⌬CTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4 At 's CTD to the VirE2 effector, although other VirD4 At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.T he type IV secretion systems (T4SSs) display striking versatility among the known bacterial translocation systems in their capacity to translocate DNA and protein substrates to bacterial or eukaryotic target cells (1, 2). Members of one major T4SS subfamily, the conjugation machines, mediate transfer of mobile DNA elements and are responsible for widespr...

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“…VirD2 consists of an N-terminal relaxase domain and a large domain of unknown function (DUF) that is less conserved between different VirD2 proteins (Howard et al, 1992;van Kregten et al, 2009). To investigate whether the relaxase domain or the DUF was responsible for the interaction with histones, BiFC experiments were performed with VirD2-204…”

Section: Resultsmentioning

confidence: 99%

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

et al. 2015

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Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 59 end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His 6 -tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA.

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Agrobacterium-Mediated T-DNA Transfer and Integration by Minimal VirD2 Consisting of the Relaxase Domain and a Type IV Secretion System Translocation Signal

Cited by 47 publications

References 45 publications

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

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