TY - JOUR A1 - Beitzinger, Christoph A1 - Bronnhuber, Annika A1 - Duscha, Kerstin A1 - Riedl, Zsuzsanna A1 - Huber-Lang, Markus A1 - Benz, Roland A1 - Hajos, György A1 - Barth, Holger T1 - Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin JF - PLoS ONE N2 - 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. KW - intoxication KW - chloroquine KW - toxins KW - anthrax KW - cell membranes KW - lipid bilayer KW - macrophages KW - membrane potential Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130097 VL - 8 IS - 6 ER - TY - JOUR A1 - Beitzinger, Christoph A1 - Stefani, Caroline A1 - Kronhardt, Angelika A1 - Rolando, Monica A1 - Flatau, Gilles A1 - Lemichez, Emanuel A1 - Benz, Roland T1 - Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA) N2 - It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His6-tag to different proteins increased their binding affinity to the protective antigen (PA) PA63-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB7/8-family of toxins. His6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to 270 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA63. Our results revealed that addition of His6-tags to several factors increase their binding properties to PA63 and enhance the property to intoxicate cells. KW - Biologie Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76325 ER - TY - JOUR A1 - Kronhardt, Angelika A1 - Beitzinger, Christoph A1 - Barth, Holger A1 - Benz, Roland T1 - Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells JF - Toxins N2 - 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. KW - C2-toxin KW - iota-toxin KW - binding components KW - chloroquine KW - black lipid bilayer KW - aminoquinolinium salts Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168286 VL - 8 IS - 8 ER -