MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
MB-JASS 2007 – Session III
“Properties of Channels Formed by Bacterial Porins and Toxins”
March 11.-21. 2007 – Moscow, Russia
“Binary Bacterial Toxins – C2- and VIP-Toxin”
Michael Leuber, Tobias Neumeyer & Roland Benz
Department of Biotechnology, University Würzburg, Germany
Clostridium botulinum C2-toxin:
Clostridium botulinum C2-toxin belongs to the family of binary toxins of the A-B type.
C2-toxin consists of 2 distinct components, a component A, which is the
enzymatically active subunit C2I and a component B (C2II), which is involved in
transport of the enzymatic component into the cytosol across the target cell
membrane. There C2I exerts its catalytic activity (Considine et al, 1991; Aktories et
al, 1992; Barth et al, 2002). Other members of the family of binary toxins are iota
toxin of Clostridium perfringens (Simpson et al, 1987, Schering et al, 1988), ADPribosyltransferase of Clostridium difficile (Popoff et al, 1988; Perelle et al, 1997;
Gülke et al, 2001), Clostridium spiroforme toxin (Popoff et al, 1988; Simpson et al,
1989), anthrax toxin of Bacillus anthracis (Mock et al, 2001; Collier et al, 2003) and
the vegetative insecticidal proteins (VIPs) of Bacillus cereus and Bacillus
thuringiensis (Han et al, 1999; Leuber et al, 2006). C2I and C2II are released
separately into the extracellular media (Ohishi et al, 1980, Aktories et al, 1992). C2I
(50kDa), ADP-ribosylates monomeric G-actin but not oligomeric F-actin at position
arginine 177, which inhibits polymerization of G-actin (Aktories et al, 1986;
Vandekerckhove et al, 1988) and largely affects ATP-binding and ATPase activity
(Geipel et al, 1989). Through the action of C2I the actin cytoskeleton breaks down,
leading to cell rounding and cell death (Reuner et al, 1987; Wiegers et al, 1991). C2II
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
(80kDa), the B subunit, needs to be cleaved by trypsin to obtain its biological activity
(Ohishi, 1987). This cleavage generates a 60kDa fragment, which forms a ringshaped heptamer (Barth et al, 2000), and a 20kDa fragment, which dissociates from
C2II. The heptamer binds to a N-linked complex carbohydrate on the surface of
target cells (Eckhardt et al, 2000) and is also able to bind the enzymatic component
C2I (Ohishi et al, 1992; Barth et al, 1998; Blöcker et al, 2003). The complex is
endocytosed into the cell. Acidification of the endosome triggers C2I translocation
into the cytosol (Simpson, 1989; Barth et al, 2000). Addition of activated C2II to
artificial lipid bilayer membranes (Benz et al, 1978; Benz et al, 1979) results in
formation of ion permeable channels that are formed by C2II heptamers (Schmid et
al, 1994; Barth et al, 2000; Bachmeyer et al, 2001). These channels could serve as
translocation pathways for C2I through the endosomal membrane (Barth et al, 2000).
Evidence has been presented that the protective antigen (PA) of anthrax toxin
homologous to C2II provides such a pathway to carry the anthrax enzymatic
components (EF, LF) into the cytosol of the target cells (Zhang et al, 2004). The C2II
channel is cation-selective and voltage-gated (Schmid et al, 1994). The C2II
heptamer inserts oriented in the membrane when added to one side of the
membrane (Bachmeyer et al, 2001). Chloroquine and other 4-aminoquinolones are
able to block reconstituted C2II channels in vivo and in vitro (Bachmeyer et al, 2001).
The half saturation constant, KS, of binding of chloroquine is in the micromolar range.
The exact mechanism of intoxication as well as inhibition of channel function with 4aminoquinolones is not well understood. To localize the binding site of chloroquine,
which is presumably the same as for C2I sequence comparisons were performed of
binding components C2II, PA, iota b, and VIP1 (Ac, Bacillus thuringiensis HD201; Aa,
Bacillus cereus AB78) of the binary toxins C2, anthrax, iota and the vegetative
insecticidal proteins, respectively. The sequences of these binding components are
significantly homologous, but they differ in binding affinity for chloroquine and related
compounds (Bachmeyer et al, 2001; Knapp et al, 2002, Orlik et al, 2005; Leuber et
al, 2006). Chloroquine binds to the PA channel with much higher affinity than to C2II,
but it has a low affinity for binding to iota b and does not bind to VIP1Ac. Because of
the ionic-strength dependence of chloroquine binding to the C2II heptamer
(Bachmeyer et al, 2001) and the fact that this drug has two positive charges at
neutral pH, negatively charged amino acids, which are present in C2II or PA but not
in iota b or VIP1Ac/Aa, could represent putative sites for binding. Possible candidates
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
are E399 and D426. The C2II heptamer is not known in its membrane-spanning
structure, which applies also to the structure of PA. However, in the case of the PA a
water-soluble precursor (prepore) has been crystallized (Petosa et al, 1997). Using
the structure of the PA prepore and the sequence comparisons mentioned above, we
identified some negatively charged amino acids as putative candidates for the
interaction with 4-aminoquinolones (E399, D426). These amino acids are presumably
located in the lumen of the channel. Furthermore, residue F427 of PA (also known as
Φ-clamp) plays a crucial role for translocation of EF or LF into the cytosol and
influences also binding of quinacrine (Krantz et al, 2005). C2II has also a
phenylalanine in a relevant position (F428). To check its role in the interaction of C2II
with 4-aminoquinolones F428A was mutated in several amino acids with different or
similar properties (F428A, F428D, F428Y, F428W). Here we show that residues
E399, D426 and F428 are important for binding of 4-aminoquinolones, which blocks
the C2II channels. Mutation of these residues leads to altered channel properties
such as single channel conductance, ion selectivity, voltage-dependence, and ionicstrength dependence of inhibitor binding. E399 and D426 are also relevant for
assembly of the channel, membrane insertion and channel function.
Bacillus thuringiensis VIP-Toxin:
The Gram-positive bacterium Bacillus thuringiensis is a common soil microorganism
that has been used as a biological pesticide for several decades. Its insecticidal
activity is mainly contributed to parasporal crystalline δ-endotoxin inclusions, formed
during sporulation (Schnepf et al, 1998), and to some extent to the vegetative
insecticidal proteins, the VIP-Toxins (Warren, 1997; Han et al, 1999). The detailed
molecular mechanisms mediating the insecticidal activity of Bacillus-produced δendotoxins have been described as a multistep process, which initiates upon
ingestion of the protein crystals. The insecticidal δ-endotoxins are solubilized in the
insect midgut and subsequently undergo site-specific proteolysis from their N- and Cterminus. These activated polypeptides bind to receptors in the midgut epithelium
and form ion channels, inducing osmotic lysis of the epithelium cells and subsequent
death of the larvae (Lorence et al, 1995). Beside these crystalline proteins B.
thuringiensis produces two known classes of vegetative expressed insecticidal
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
proteins. These include the binary toxins Vip1 and Vip2 with coleopteran specificity
and Vip3 exhibiting lepidopteran specificity (Warren 1997; Estruch et al, 1997; Lee et
al, 2003). Vip-toxins, so called because of their production during vegetative growth
phase extent their insecticidal properties especially against the Western Corn
Rootworm (Diabrotica virgifera virgifera LeConte) and the European Corn Borer
(Ostrinia nubilalis), both widespread corn pests, less affected by the δ-Endotoxins
(Warren, 1997). The binary Vip-toxin produced by B. thuringiensis HD201 belongs to
the group of A-B toxins with an A moiety (Vip2Ac) for the enzymatic reaction, and the
B moiety (Vip1Ac) responsible for the translocation of component A across the cell
membrane (Barth et al, 2004). Both polypeptides function separately, with the
membrane-binding 66 kDa Vip1Ac creating the pathway into the cytoplasm for the 45
kDa Vip2Ac, a NAD-dependent ADP-ribosyltransferase which likely targets Arginine
177 of monomeric G-actin, but not polymerized F-actin, hereby disrupting the integrity
of the cytoskeleton, resulting in a rounding-up and death of the target cells (Han et al,
1999; Aktories and Wegner, 1992). Both δ-endotoxins and vegetative insecticidal
proteins are currently used in different forms of commercially available biopesticides
for agriculture and mosquito control (Warren et al, 2000).
Vip1Ac was obtained in its native form from a Bacillus culture supernatant as well as
from heterologous expression in E. coli. Purified, native VIP1Ac forms well defined
channels exhibiting two conductance states at higher salt concentration (Leuber et al,
2006). The addition of the protein extract to the aqueous phase (concentration about
10ng/ml) bathing a black lipid bilayer resulted in a very fast reconstitution of channels
(Benz et al, 1978; Benz et al, 1979). Single channel analysis revealed two
conductance states of about 350 pS and 700 pS in 1M KCl, 10mM MES, pH 5.5
(applied voltage 20mV). Interestingly, these two conductance states were only visible
at high electrolyte concentration. At salt concentrations below 500 mM KCl only one
conductance state was observed. The two states could be explained by the formation
of two different types of oligomers. Alternatively it is also possible that at high ionic
strength two channels were frequently formed at once because the higher
conductance steps were exactly twice that of the smaller ones. The data also indicate
that the single-channel conductance was approximately a linear function of the bulk
aqueous KCl concentration (Leuber et al, 2006).
The selectivity of the Vip1Ac channel was determined in zero-current membrane
potential measurements in the presence of salt gradients. After incorporation of a
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
large number of channels in membranes bathed in 100 mM KCl, 5-fold salt gradients
were established across the membranes by the addition of small amounts of
concentrated KCl solution to one side of the membrane. In all cases, the more diluted
side of the membrane became negative (on average -12.5 mV), which indicated
preferential movement of anions through the Vip1Ac channel, i.e. it is anion-selective.
This result is consistent with the single channel conductance data where anions had
a somewhat stronger influence on the single channel conductance of Vip1Ac as
compared to cations (Leuber et al, 2006). Analysis of the zero-current membrane
potentials using the Goldman-Hodgkin-Katz equation showed that the permeability
ratio Pcation over Panion was about 0.36.
In single-channel recordings native Vip1Ac exhibited significant channel flickering at
higher positive voltages applied to the cis-side (side of protein addition), meaning it
showed rapid transitions between open and closed configuration. This could be
caused by voltage-dependent closing of the channel, and therefore we increased in
single- and multichannel experiments the applied trans-membrane voltage. Native
Vip1Ac was added in a concentration of ~10 ng/ml to one side of a black diphytanoyl
phosphatidylcholine/n-decane membrane (the cis-side), and the conductance
increase was followed for about 25 min until stationary phase was reached. At this
point, we applied different positive and negative potentials (with respect to the cisside) to the membrane starting from 20 mV. Then we repeated the experiment with
increasing potentials. After application of +90 mV to the cis-side of the membrane a
stepwise decrease of the membrane current could be observed, indicating the
complete closing of the channel at high positive potentials. For negative potentials at
the cis-side, the current did not decrease even when the membrane potential was as
high as 120 mV. This result indicated asymmetric insertion of Vip1Ac into the
membranes (Leuber et al, 2006). The addition of protein to both sides of the
membrane resulted in a symmetric response to the applied voltage. The results
indicated full orientation of the Vip1Ac channel when the protein was added only to
one side of the membrane. This result is in clear contrast to the observed voltage
dependency of other related channel forming toxins like Iota-toxin (Ib), C2-toxin (C2II) and Anthrax toxin (PA) where channel closure is observed at high negative
voltages applied to the cis-side of the membrane (Bachmeyer et al, 2001; Knapp et
al, 2002; Orlik et al, 2005).
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
References
Aktories, K., and Wegner, A. (1992). Mechanisms of the cytopathic action of actin-ADP-ribosylating
toxins. Mol. Microbiol. 6: 2905-2908.
Aktories, K., Wille, M., and Just, I. (1992). Clostridial actin-ADP-ribosylating toxins. Curr. Top.
Microbiol. Immunol. 175: 97-113.
Aktories, K., Bärmann, M., Ohishi, I., Tsuyama, S., Jakobs, K. H., and Habermann, E. (1986).
Botulinum C2 toxin ADP-ribosylates actin. Nature 322: 390-392.
Bachmeyer, C., Benz, R., Barth, H., Aktories, K., Gilbert, M., and Popoff, M. R. (2001). Interaction of
Clostridium botulinum C2-toxin with lipid bilayer membranes and Vero-cells: inhibition of
channel function by chloroquine and related compounds in vitro and intoxification in vivo.
FASEB J. 15: 1658-1660.
Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998). The N-terminal part of the enzyme
component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding
component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion
toxin. Infect. Immun. 66: 1364-1369.
Barth, H., Blöcker, D., Behlke, J., Bergsma-Schutter, W., Brisson, A., Benz, R. and Aktories, K. (2000).
Cellular uptake of Clostridium botulinum C2 toxin requires oligomerization and acidification. J.
Biol. Chem. 275: 18704-18711.
Barth, H., Blöcker, D., and Aktories, K. (2002). The uptake machinery of clostridial actin ADPribosylating toxins: a cell delivery system for fusion proteins and polypeptide drugs. Naunyn
Schmiedebergs Arch. Pharmacol. 366: 501-512.
Barth, H., Aktories, K., Popoff, M. R., and Stiles, B. G. (2004). Binary bacterial toxins: biochemistry,
biology, and applications of common Clostridium and Bacillus proteins. Microbiol. Mol. Biol.
Rev. 68: 373-402.
Benz, R., Janko, K., Boos, W., and Läuger, P. (1978). Formation of large, ion-permeable membrane
channels by the matrix protein (porin) of Escherichia coli. Biochim. Biophys. Acta 511: 305319.
Benz, R., Janko, K., and Läuger, P. (1979). Ionic selectivity of pores formed by the matrix protein
(porin) of Escherichia coli. Biochim. Biophys. Acta 551: 238-247.
Blöcker, D., Bachmeyer, C., Benz, R., Aktories, K., and Barth, H. (2003). Channel Formation by the
Binding Component of Clostridium botulinum C2 Toxin: Glutamate 307 of C2II Affects Channel
Properties in Vitro and pH-Dependent C2I Translocation in Vivo. Biochemistry 42: 5368-5377.
Collier, R. J., and Young, J. A. (2003). Anthrax toxin. Annu. Rev. Cell Dev. Biol. 19: 45-70.
Considine, R. V., and Simpson, L. L. (1991). Cellular and molecular actions of binary toxins
possessing ADP-ribosyltransferase activity. Toxicon 29: 913-936.
Eckhardt, M., Barth, H., Blöcker, D., and Aktories, K. (2000). Binding of Clostridium botulinum C2 toxin
to asparagine-linked complex and hybrid carbohydrates. J. Biol. Chem. 275: 2328-2334.
Estruch, J. J., Warren, G. W., Mullins, M. A., Nye, G. J., Craig, J. A., and Koziel, M. G. (1996). Vip3A,
a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities
against lepidopteran insects. Proc. Natl. Acad. Sci. USA, 93: 5389-5394.
Geipel, U., Just, I., Schering, B., Haas, D., and Aktories, K. (1989). ADP-ribosylation of actin causes
increase in the rate of ATP exchange and inhibition of ATP hydrolysis. Eur. J. Biochem. 179:
229-232.
Gülke, I., Pfeifer, G., Liese, J., Fritz, M., Hofmann, F., Aktories, K., and Barth, H. (2001).
Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from
Clostridium difficile. Infect. Immun. 69: 6004-6011.
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
Han, S., Craig, J. A., Putnam, C. D., Carozzi, N. B., and Tainer, J. A. (1999). Evolution and
mechanism from structures of an ADP-ribosylating toxin and NAD-complex. Nat. Struct. Biol.
6: 932-936.
Knapp, O., Benz, R., Gibert, M., Marvaud, J. C., and Popoff, M. R. (2002). Interaction of Clostridium
perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the
activated binding component Ib and channel block by the enzyme component Ia. J. Biol.
Chem. 277: 6143-52.
Krantz, B. A., Melnyk, R. A., Zhang, S., Juris, S. J., Lacy, D. B., Wu, Z., Finkelstein, A., and Collier, R.
J. (2005). A phenylalanine clamp catalyzes protein translocation through the anthrax toxin
pore. Science 309: 777-81.
Lee, M. K., Walters, F. S., Hart, H., Palekar, N., and Chen J. S. (2003). The mode of action of the
Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab deltaendotoxin. Appl. Environ. Microbiol. 69: 4648-4657.
Leuber, M., Orlik, F., Schiffler, B., Sickmann, A., and Benz, R. (2006). Vegetative insecticidal protein
(Vip1Ac) of Bacillus thuringiensis HD201: Evidence for oligomer and channel formation.
Biochemistry 45: 283-288.
Lorence, A. A., Darzon, C., Diaz, A., Lievano, R., Quintero, R., and Bravo, A. (1995). Delta-endotoxins
induce cation channels in Spodoptera frugiperda brush border membranes in suspension and
in planar lipid bilayers. FEBS Lett. 360: 217–222.
Mock, M., and Fouet, A. (2001). Anthrax. Annu. Rev. Microbiol. 55: 647-671.
Ohishi, I., Iwasaki, M., and Sakaguchi, G. (1980). Purification and characterization of two components
of botulinum C2 toxin. Infect. Immun. 30: 668-673.
Ohishi, I. (1987). Activation of botulinum C2 toxin by trypsin. Infect. Immun. 55: 1461-1465.
Ohishi, I., and Yanagimoto, A. (1992). Visualizations of binding and internalization of two nonlinked
protein components of C. botulinum C2 toxin in tissue culture cells. Infect. Immun. 60: 46484655.
Orlik, F., Schiffler, B., and Benz, R (2005). Anthrax toxin protective antigen: inhibition of channel
function by chloroquine and related compounds and study of binding kinetics using the current
noise analysis. Biophys J. 88: 1715-24.
Perelle, S., Gibert, M., Bourlioux, P., Corthier, G., and Popoff, M.R. (1997). Production of a complete
binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infect.
Immun. 65: 1402-1407.
Petosa, C., Collier, R. J., Klimpel, K. R., Leppla, S. H., and Liddington, R. C. (1997). Crystal structure
of the anthrax toxin protective antigen. Nature 385: 833-838.
Popoff, M. R., Rubin, E. J., Gill, D. M., and Boquet (1988). Actin-specific ADP-ribosyltransferase
produced by a Clostridium difficile strain. Infect. Immun. 56: 2299-2306.
Popoff, M. R., and Boquet , P. (1988b). Clostridium spiroforme toxin is a binary toxin which ADPribosylates cellular actin. Biochem. Biophys. Res. Commun. 152: 1361-1368.
Reuner, K. H., Presek, P., Boschek, C. B., and Aktories, K. (1987). Botulinum C2 toxin ADPribosylates actin and disorganizes the microfilament network in intact cells. Eur. J. Cell Biol.
43: 134-140.
Schering, B., Bärmann, M., Chhatwal, G. S., Geipel, U., and Aktories, K. (1988). ADP-ribosylation of
skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin. Eur. J. Biochem.
171: 225-229.
Schmid, A., Benz, R., Just, I., and Aktories, A. (1994). Interaction of Clostridium botulinum C2 toxin
with Lipid Bilayer Membranes. J. Biol. Chem. 269: 16706-16711.
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., and Dean,
D. H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev.
62: 775-806.
Simpson, L. L., Stiles, B. G., Zepeda, H. H., and Wilkins, T. D. (1987). Molecular basis for the
pathological actions fo Clostridium perfringens iota toxin. Infect. Immun. 55: 118-122.
MB-JASS 2007 – Session III – Properties of Channels Formed by Bacterial Porins and Toxins
Simpson, L. L., Stiles, G. B., Zepeda, H., and Wilkins, T. D. (1989). Production by Clostridium
spiroforme of an iota-like toxin that possesses mono(ADP-ribosyl)transferase activity:
identification of a novel class of ADP-ribosyltransferases. Infect. Immun. 57: 255-261.
Vandekerckhove, J., Schering, B., Bärmann, M., and Aktories, K. (1988). Botulinum C2 toxin ADPribosylates cytoplasmic β/ν-actin in arginine 177. J. Biol. Chem. 263: 696-700.
Warren, G. W. (1997). Advances in Insect Control, the Role of Transgenic Plants. Taylors & Francis
Ltd, London.
Warren, G. W., Koziel, M. G., Mullins, M. A., Nye, G. J., Carr, B., Desai, N. M., and Kostichka, K.
(2000). Genes encoding insecticidal proteins. United States Patent: 6,066,783.
Wiegers, W., Just, I., Müller, H., Hellwig, A., Traub, P., and Aktories, K. (1991). Alteration of the
cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3
ADP-ribosyltransferase. Eur. J. Cell Biol. 54: 237-245.
Zhang, S., Finkelstein, A., and Collier, R. J. (2004). Evidence that translocation of anthrax toxin´s
lethal factor is initiated by entry of its N terminus into the protective antigen channel. PNAS
101: 16756-16761.
Zhang, S.,Udho, E., Wu, Z., Collier, R. J., and Finkelstein, A. (2004). Protein Translocation through
Anthrax Toxin Channels Formed in Planar Lipid Bilayers. Biophysical Journal 87: 3842-3849.