Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli–Bartonella spp. shuttle cloning vector

Seubert, Anja; Falch, Christine; Birtles, Richard J.; Schülein, Ralf; Dehio, Christoph

doi:10.1016/s0147-619x(02)00103-8

Cited by 21 publications

(20 citation statements)

References 35 publications

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“…For construction of pRS117, the 1861 bp HincII-AflIII fragment of pWay19 (gift from the Molecular Motion Laboratory, Montana State University, Bozeman, MT) containing the egfp gene under the control of the CMV promoter was blunt-ended by using Klenow polymerase and ligated with HincII cleaved pBGR-K18, a derivative of pBGR1 (20). The plasmid containing the egfp gene and the kan gene in the same orientation was selected, yielding pRS117.…”

Section: Methodsmentioning

confidence: 99%

“…For construction of pRS122, a 567-bp fragment encoding the secretion signal of B. henselae BepD flanked by two AgeI restriction sites, obtained by using pRS51 as template and the oligonucleotides prRS350 and prRS351 (Table S3), was ligated with pRS117, and the plasmid containing the fragment in the correct orientation resulting in a fusion with the mob gene was selected. For construction of pRGS06, pCHF01 (20) was digested with SalI/XmaI/NcoI and the 2,541-bp SalI-XmaI fragment containing the disrupted mob gene was ligated with pRS122 (SalI/XmaI). pRS130 was obtained by introducing the 1514 bp SnaBI-EcoRV fragment of pRS56 (19) containing the neo gene into HincIIcleaved pRS129.…”

Section: Methodsmentioning

confidence: 99%

“…To test whether the VirB/VirD4 T4SS of B. henselae may-in addition to its role in intracellular protein delivery-be capable of mobilizing DNA into human cells, we took advantage of the Bartonella-specific cryptic plasmid pBGR1, which, as its only discernable genetic elements, encodes a replication protein (Rep) and respective origin of replication (oriV), as well as a relaxase (Mob) and respective oriT (20). Although the Mob relaxase of pBGR1 does not contain a recognizable BID-like domain, we wondered whether this naturally occurring mobilizable plasmid might transfer into human cells during the course of infection.…”

Section: B Henselaementioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: B Henselaementioning

confidence: 99%

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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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“…In contrast to the VirB/D4 T4SS of Bartonella, the Trw T4SS apparently neither harbors a coupling protein nor translocates any known effectors, but it carries numerous tandem gene duplications coding mostly for surface-exposed subunits (401). Coduplicated gene copies encoding a core subunit are highly conserved, while the genes encoding putative minor and major pilus subunits have been shaped by diversifying selection.…”

Section: Infection Of Erythrocytesmentioning

confidence: 99%

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

2012

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SUMMARY Bartonella spp. are facultative intracellular pathogens that employ a unique stealth infection strategy comprising immune evasion and modulation, intimate interaction with nucleated cells, and intraerythrocytic persistence. Infections with Bartonella are ubiquitous among mammals, and many species can infect humans either as their natural host or incidentally as zoonotic pathogens. Upon inoculation into a naive host, the bartonellae first colonize a primary niche that is widely accepted to involve the manipulation of nucleated host cells, e.g., in the microvasculature. Consistently, in vitro research showed that Bartonella harbors an ample arsenal of virulence factors to modulate the response of such cells, gain entrance, and establish an intracellular niche. Subsequently, the bacteria are seeded into the bloodstream where they invade erythrocytes and give rise to a typically asymptomatic intraerythrocytic bacteremia. While this course of infection is characteristic for natural hosts, zoonotic infections or the infection of immunocompromised patients may alter the path of Bartonella and result in considerable morbidity. In this review we compile current knowledge on the molecular processes underlying both the infection strategy and pathogenesis of Bartonella and discuss their connection to the clinical presentation of human patients, which ranges from minor complaints to life-threatening disease.

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“…The start codon of both downstream ORFs was separated from the termination codon of the preceding ORF-2 by a three-nucleotide gap (of GGC) and thus remained in frame. ORF-2 (nucleotides 2441 -3016) encodes a 191 a.a. (21.5 kDa) protein with the best BLASTX hit of 60% identity to the plasmid-encoded replication protein, repA (YP_133706) of Bartonella grahamii plasmid pBGR1 (Seubert et al, 2003). The predicted products of ORF-3 and ORF-4, which encodes 64 a.a. (7.4 kDa) and 114 a.a (13.0 kDa) proteins, showed no significant similarity to any known protein sequences.…”

Section: Results and Discussion 31 Isolation And Characterisation Ofmentioning

confidence: 99%

The genetic toolbox for Acidovorax temperans

Boycheva

Pichler

Heijstra

et al. 2015

Journal of Microbiological Methods

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Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli–Bartonella spp. shuttle cloning vector

Cited by 21 publications

References 35 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

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

The genetic toolbox for Acidovorax temperans

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