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  • 1
    Online Resource
    Online Resource
    A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells
    Proceedings of the National Academy of Sciences ; 2005
    In:  Proceedings of the National Academy of Sciences Vol. 102, No. 3 ( 2005-01-18), p. 856-861
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 102, No. 3 ( 2005-01-18), p. 856-861
    Abstract: Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella -translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella -specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0406796102
    RVK:
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2005
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  • 2
    Online Resource
    Online Resource
    Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)
    Proceedings of the National Academy of Sciences ; 2021
    In:  Proceedings of the National Academy of Sciences Vol. 118, No. 12 ( 2021-03-23)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 118, No. 12 ( 2021-03-23)
    Abstract: Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2023245118
    RVK:
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2021
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    detail.hit.zdb_id: 1461794-8
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  • 3
    Online Resource
    Online Resource
    Splicing of the rolA Transcript of Agrobacterium rhizogenes in Arabidopsis
    American Association for the Advancement of Science (AAAS) ; 1994
    In:  Science Vol. 266, No. 5193 ( 1994-12-23), p. 1986-1988
    Details
    In: Science, American Association for the Advancement of Science (AAAS), Vol. 266, No. 5193 ( 1994-12-23), p. 1986-1988
    Type of Medium: Online Resource
    ISSN: 0036-8075 , 1095-9203
    URL: Article
    DOI: 10.1126/science.7528444
    RVK:
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    Language: English
    Publisher: American Association for the Advancement of Science (AAAS)
    Publication Date: 1994
    detail.hit.zdb_id: 128410-1
    detail.hit.zdb_id: 2066996-3
    detail.hit.zdb_id: 2060783-0
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  • 4
    Online Resource
    Online Resource
    Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens
    Proceedings of the National Academy of Sciences ; 2014
    In:  Proceedings of the National Academy of Sciences Vol. 111, No. 12 ( 2014-03-25), p. 4548-4553
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 111, No. 12 ( 2014-03-25), p. 4548-4553
    Abstract: Systematic genetic perturbation screening in human cells remains technically challenging. Typically, large libraries of chemically synthesized siRNA oligonucleotides are used, each designed to degrade a specific cellular mRNA via the RNA interference (RNAi) mechanism. Here, we report on data from three genome-wide siRNA screens, conducted to uncover host factors required for infection of human cells by two bacterial and one viral pathogen. We find that the majority of phenotypic effects of siRNAs are unrelated to the intended “on-target” mechanism, defined by full complementarity of the 21-nt siRNA sequence to a target mRNA. Instead, phenotypes are largely dictated by “off-target” effects resulting from partial complementarity of siRNAs to multiple mRNAs via the “seed” region (i.e., nucleotides 2–8), reminiscent of the way specificity is determined for endogenous microRNAs. Quantitative analysis enabled the prediction of seeds that strongly and specifically block infection, independent of the intended on-target effect. This prediction was confirmed experimentally by designing oligos that do not have any on-target sequence match at all, yet can strongly reproduce the predicted phenotypes. Our results suggest that published RNAi screens have primarily, and unintentionally, screened the sequence space of microRNA seeds instead of the intended on-target space of protein-coding genes. This helps to explain why previously published RNAi screens have exhibited relatively little overlap. Our analysis suggests a possible way of identifying “seed reagents” for controlling phenotypes of interest and establishes a general strategy for extracting valuable untapped information from past and future RNAi screens.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1402353111
    RVK:
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2014
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  • 5
    Online Resource
    Online Resource
    Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice
    Proceedings of the National Academy of Sciences ; 2022
    In:  Proceedings of the National Academy of Sciences Vol. 119, No. 25 ( 2022-06-21)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 119, No. 25 ( 2022-06-21)
    Abstract: The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti- Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella -neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella -neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2202059119
    RVK:
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2022
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  • 6
    Online Resource
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    Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae
    Proceedings of the National Academy of Sciences ; 2011
    In:  Proceedings of the National Academy of Sciences Vol. 108, No. 35 ( 2011-08-30), p. 14643-14648
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 35 ( 2011-08-30), p. 14643-14648
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1019074108
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2011
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  • 7
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    Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification
    Proceedings of the National Academy of Sciences ; 2016
    In:  Proceedings of the National Academy of Sciences Vol. 113, No. 5 ( 2016-02-02)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 113, No. 5 ( 2016-02-02)
    Abstract: Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (α inh ) that partly obstructs the active site. For the single-domain class III Fic proteins, the α inh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis , thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1516930113
    RVK:
    TA 1000
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2016
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  • 8
    Online Resource
    Online Resource
    Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production
    Proceedings of the National Academy of Sciences ; 2012
    In:  Proceedings of the National Academy of Sciences Vol. 109, No. 24 ( 2012-06-12), p. 9581-9586
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 24 ( 2012-06-12), p. 9581-9586
    Abstract: Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: ( i ) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli ; ( ii ) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; ( iii ) surface plasmon resonance experiments; and ( iv ) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1117651109
    RVK:
    TA 1000
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    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2012
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