@article{DmochewitzFoertschZwergeretal.2013,
  author    = {Dmochewitz, Lydia and F{\"o}rtsch, Christina and Zwerger, Christian and Vaeth, Martin and Felder, Edward and Huber-Lang, Markus and Barth, Holger},
  title     = {A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages},
  series = {PLoS ONE},
  volume    = {8},
  journal   = {PLoS ONE},
  number    = {1},
  doi       = {10.1371/journal.pone.0054517},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131189},
  pages     = {e54517},
  year      = {2013},
  abstract  = {Background: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. Methodology/Principal Findings: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (~50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. Conclusions/Significance: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.},
  language  = {en}
}
@article{BeitzingerBronnhuberDuschaetal.2013,
  author    = {Beitzinger, Christoph and Bronnhuber, Annika and Duscha, Kerstin and Riedl, Zsuzsanna and Huber-Lang, Markus and Benz, Roland and Hajos, Gy{\"o}rgy and Barth, Holger},
  title     = {Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin},
  series = {PLoS ONE},
  volume    = {8},
  journal   = {PLoS ONE},
  number    = {6},
  doi       = {10.1371/journal.pone.0066099},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130097},
  pages     = {e66099},
  year      = {2013},
  abstract  = {Background Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric \(PA_{63}\) binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the \(PA_{63}\)-channel in a dose dependent way. Methodology/Principal Findings Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the \(PA_{63}\)-channel in the µM range, when both, inhibitor and \(PA_{63}\) are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of \(PA_{63}\)-channel function also efficiently block intoxication of the cells by the combination lethal factor and \(PA_{63}\) in the same concentration range as they block the channels in vitro. Conclusions/Significance These results strongly argue in favor of a transport of lethal factor through the \(PA_{63}\)-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.},
  language  = {en}
}
@article{KronhardtBeitzingerBarthetal.2016,
  author    = {Kronhardt, Angelika and Beitzinger, Christoph and Barth, Holger and Benz, Roland},
  title     = {Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells},
  series = {Toxins},
  volume    = {8},
  journal   = {Toxins},
  number    = {8},
  doi       = {10.3390/toxins8080237},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168286},
  pages     = {237},
  year      = {2016},
  abstract  = {C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.},
  language  = {en}
}