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. 2011 Nov;193(22):6257-65.
doi: 10.1128/JB.05905-11. Epub 2011 Sep 9.

Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System Into Both Bacteria and Human Cells

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Free PMC article

Transfer of R388 Derivatives by a Pathogenesis-Associated Type IV Secretion System Into Both Bacteria and Human Cells

Esther Fernández-González et al. J Bacteriol. .
Free PMC article

Abstract

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.

Figures

Fig. 1.
Fig. 1.
(A) Plasmids used to test DNA transfer into human cells. These plasmids contain the pBBR6 replication origin, a gentamicin resistance gene, the indicated R388 Dtr region (oriT, trwA, trwB, trwC), and a eukaryotic egfp expression cassette (PCMV-egfp-SV40 polyadenylation signal). (B) Western blots with anti-TrwB (top) and anti-TrwC (bottom) primary antibodies to detect TrwB and TrwC steady-state levels in E. coli carrying plasmids coding for the indicated R388 proteins. Black arrows point to TrwB, TrwC, and TrwC-BID; the gray arrow indicates a unspecific band detected by the anti-TrwB antibody (10).
Fig. 2.
Fig. 2.
Fluorescence-activated cell sorting (FACS) graph plotting cell granularity (side scatter A [SSC-A]) versus eGFP fluorescence intensity (in abscissas). (A) Uninfected cells, which determined eGFP background. The square marks the population considered positive. (B) Cells infected by B. henselae containing plasmid pHP161 (oriT+trwABC).
Fig. 3.
Fig. 3.
Number of intracellular bacteria recovered from infected EA.hy926 cells after 24, 48, and 72 h postinfection.
Fig. 4.
Fig. 4.
Percentage of GFP-positive cells infected by the indicated B. henselae mutant strains carrying plasmid pHP161 (oriT trwABC) (panel A) or wild-type B. henselae carrying plasmids which expressed the indicated TrwB or TrwC variants (panel B). The bars represent means with standard deviation from at least three independent experiments done in triplicate. Strains in panel A are identified by the deleted trw-Bt or vir genes, e.g., the ΔB2-11 is the strain with a virB2-to-virB11 deletion, and so on. Plasmids in panel B are named by their difference from pHP161, containing wild-type trwABC genes, as follows: ABC, wild-type plasmid; ΔB and ΔC, deletion of trwB and trwC, respectively; C::BID, TrwC-BID fusion protein; TrwB and TrwC point mutants are indicated.
Fig. 5.
Fig. 5.
Bacterial two-hybrid assay used to detect protein-protein interactions. Pairs of plasmids encoding fusions of the indicated proteins to the T18 and T25 domains of adenylate cyclase were transformed into test strain DHM1. Transformants were streaked on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing plates. Blue color reflects interaction between the fused proteins. C+ and C−, plasmids pT25zip + pUT18Czip and pUT18C, respectively. Abbreviations for fused proteins: B, R388 TrwB; point TrwB mutations are indicated in parentheses; E, TrwE-Bt; B10, B. henselae VirB10; D4, B. henselae VirD4.

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