Chloroquine-analogues block anthrax protective antigen channels in steady-state and kinetic studies.

The tripartite anthrax toxin from Bacillus anthracis represents the prototype of A-B type of toxins, where the effector A (an enzymatic subunit) is transported with the help of a binding component B into a target cell. Anthrax toxin consists of three different molecules, two effectors, lethal factor (LF) and edema factor (EF) and the binding component also known as protective antigen (PA). PA forms heptamers or octamers following binding to host cell's receptors and mediates the translocation of the effectors into the cytosol via the endosomal pathway. The cation-selective PA63-channel is able to reconstitute in lipid membranes and can be blocked by chloroquine and other heterocyclic compounds. This suggests that the PA63-channel contains a binding site for quinolines. In this study, we investigated the structure-function relationship of different quinolines for the block of the PA63-channel. The affinity of the different chloroquine analogues to the PA63-channel as provided by the equilibrium dissociation constant was measured using titrations. Some quinolines had a much higher affinity to the PA63-channel than chloroquine itself. We also performed ligand-induced current noise measurements using fast Fourier transformation to get insight in the kinetics of the binding of some quinolines to the PA63-channel. The on-rate constants of ligand binding were around 108 M-1·s-1 at 150 mM KCl and were only little dependent on the individual quinoline. The off-rates varied between 4 s-1 and 160 s-1 and depended much more on the structure of the molecules than the on-rate constants. The possible use of the 4-aminoquinolines as a therapy is discussed.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells.

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.

Role of N-terminal His6-Tags in binding and efficient translocation of polypeptides into cells using anthrax protective antigen (PA).

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 AB(7/8)-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 His(6)-tag to different proteins increased their binding affinity to the protective antigen (PA) PA(63)-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB(7/8)-family of toxins. His(6)-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 -70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA(63). Cell-based experiments demonstrated that addition of an N-terminal His(6)-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA(63). Our results revealed that addition of His(6)-tags to several factors increase their binding properties to PA(63) and enhance the property to intoxicate cells.

Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells.

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore.

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.