Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker

Dehio, Michaela; Knorre, Alexander; Lanz, Christa; Dehio, Christoph

doi:10.1016/s0378-1119(98)00319-9

Cited by 68 publications

(51 citation statements)

References 14 publications

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“…B. henselae and Escherichia coli strains were grown as described (21,35). Bacterial strains used in this study and their origins are listed in Table S2.…”

Section: Methodsmentioning

confidence: 99%

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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“…B. henselae and Escherichia coli strains were grown as described (21,35). Bacterial strains used in this study and their origins are listed in Table S2.…”

Section: Methodsmentioning

confidence: 99%

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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“…E. coli DH5␣, an encapsulated K12 laboratory strain, carrying the green fluorescent protein (gfp)-mut2 gene (16), was used for phagocytosis. Bacteria were freshly grown in Lennox-L-Broth-medium (Invitrogen) until early logarithmic growth, resuspended in phosphate-buffered saline (PBS), and used immediately.…”

Section: Methodsmentioning

confidence: 99%

Diminished Phagocytosis-Induced Cell Death (PICD) in Neonatal Monocytes upon Infection with Escherichia coli

et al. 2008

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An imbalance in apoptosis or survival of immune cells plays an essential role in the pathophysiology of sepsis. Phagocytosis-induced cell death (PICD) is a common result of the pathogenhost cell interaction mediated by reactive oxygen species (ROS). Neonatal sepsis is frequently characterized by hyperinflammation. Cord blood monocytes (CBMO) are equivalent to monocytes of adults [peripheral blood monocytes (PBMO)], both in terms of phagocytosis and killing of Escherichia coli. We investigated whether CBMO are less sensitive toward PICD compared with PBMO. Monocytes were infected with green fluorescent protein (GFP)-labeled E. coli. Phagocytic activity, cell-count, Annexin V staining, hypoploid DNA content, CD95 and CD95L expression, and caspase-8 and -9 activities were analyzed by flow cytometry, ROS production by chemiluminescence, and CD95L mRNA expression by reverse-transcriptase polymerase chain reaction. With equal phagocytic activity and ROS production, PBMO cell count was decreased by 82 Ϯ 6% versus 28 Ϯ 8% for CBMO after infection. Annexin V binding was enhanced fivefold on PBMO; 56 Ϯ 15% of PBMO showed a hypodiploid DNA content compared with 9 Ϯ 6% of CBMO. Caspases CD95L and CD95L mRNA were up-regulated in PBMO. Our results indicate that CBMO are less sensitive toward E. coli-mediated PICD than PBMO. Modifying monocyte apoptosis may be a target for future interventions in sepsis. (Pediatr Res 63: [33][34][35][36][37][38] 2008)

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“…Bacterial Culture E. coli DH5α, an encapsulated K12 laboratory strain, carrying the GFP-mut2 gene (36), was used for most infection assays (E. coli-GFP) except for infection of CFSE-labeled cells, in which the wild-type E. coli DH5α (E. coli) was used. Bacteria were freshly grown in Lennox-L-Broth-medium (Invitrogen) until early logarithmic growth, resuspended in phosphate-buffered saline, and used immediately.…”

Section: Purification Of Monocytesmentioning

confidence: 99%

The CD95/CD95L pathway is involved in phagocytosis-induced cell death of monocytes and may account for sustained inflammation in neonates

et al. 2012

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Background:The propensity for sustained inflammation after bacterial infection in neonates, resulting in inflammatory sequelae such as bronchopulmonary dysplasia and periventricular leucomalacia, is well known, but its molecular mechanisms remain elusive. Termination of inflammatory reactions physiologically occurs early after removal of bacteria by phagocytosis-induced cell death (PIcD) of immune effector cells such as monocytes. PIcD from cord blood monocytes (cBMOs) was shown to be reduced as compared with that of peripheral blood monocytes (PBMOs) from adult donors in vitro. Methods: PBMOs, cBMOs, and Fas (cD95)-deficient (lpr) mouse monocytes were analyzed in an in vitro infection model using green fluorescence protein-labeled Escherichia coli (E. coli-GFP). Phagocytosis and apoptosis were quantified by flow cytometry and cD95L secretion was quantified by enzyme-linked immunosorbent assay. results: We demonstrate the involvement of the cD95/ cD95 ligand pathway (cD95/cD95L) in PIcD and provide evidence that diminished cD95L secretion by cBMOs may result in prolonged activation of neonatal immune effector cells. conclusion: These in vitro results offer for the first time a molecular mechanism accounting for sustained inflammation seen in neonates.

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Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker

Cited by 68 publications

References 14 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

Diminished Phagocytosis-Induced Cell Death (PICD) in Neonatal Monocytes upon Infection with Escherichia coli

The CD95/CD95L pathway is involved in phagocytosis-induced cell death of monocytes and may account for sustained inflammation in neonates

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