1 s2.0 S0022201112001358 Main

Review
Current models of the mode of action of Bacillus thuringiensis insecticidal

crystal proteins: A critical review
Vincent Vachon
a,b
, Raynald Laprade
a,c
, Jean-Louis Schwartz
a,b,d,
a
Groupe dtude des protines membranaires, Universit de Montral, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, Canada H3C 3J7
b
Department of Physiology, Universit de Montral, Canada
c
Department of Physics, Universit de Montral, Canada
d
Centre SVE, Department of Biology, Universit de Sherbrooke, 2500 boul. de lUniversit, Sherbrooke, Quebec, Canada J1K 2R1
a r t i c l e i n f o
Article history:
Received 21 March 2012
Accepted 3 May 2012
Available online 19 May 2012
Keywords:
Insecticidal toxins
Mode of action
Pore formation
Intracellular signaling
Models
Bacillus thuringiensis
a b s t r a c t
Bacillus thuringiensis (Bt) Cry toxins constitute the active ingredient in the most widely used biological
insecticides and insect-resistant transgenic crops. A clear understanding of their mode of action is neces-
sary for improving these products and ensuring their continued use. Accordingly, a long history of inten-
sive research has established that their toxic effect is due primarily to their ability to form pores in the
plasma membrane of the midgut epithelial cells of susceptible insects. In recent years, a rather elaborate
model involving the sequential binding of the toxins to different membrane receptors has been devel-
oped to describe the events leading to membrane insertion and pore formation. However, it was also pro-
posed recently that, in contradiction with this mechanism, Bt toxins function by activating certain
intracellular signaling pathways which lead to the necrotic death of their target cells without the need
for pore formation. Because work in this eld has largely focused, for several years, on the elaboration
and promotion of these two models, the present revue examines in detail the experimental evidence
on which they are based. It is concluded that the presently available information still supports the notion
that Bt Cry toxins act by forming pores, but most events leading to their formation, following binding of
the activated toxins to their receptors, remain relatively poorly understood.
2012 Elsevier Inc. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. The sequential binding model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Are two different receptors required? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. How critical is the removal of helix a1? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Is a toxin pre-pore required for membrane insertion? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4. Are pre-formed oligomers more efficient than toxin monomers? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. The signaling pathway model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1. Introduction
The Gram-positive spore-forming bacterium Bacillus thuringien-
sis (Bt) is characterized by its ability to form crystal-like parasporal
inclusions during sporulation (Aronson, 2002; Bulla et al., 1980;
Whiteley and Schnepf, 1986). These inclusions contain proteins,
called d-endotoxins, which are well known for their insecticidal
properties (Aronson, 1993; Aronson et al., 1986; Carlton, 1990;
Hfte and Whiteley, 1989; Lthy and Ebersold, 1981; Rogoff and
Yousten, 1969; van Frankenhuyzen, 2009). Indeed, commercial
insecticides derived from this bacterium have a long history of suc-
cessful use in the biocontrol of insect pests (Beegle and Yamamoto,
1992; Bravo et al., 2011; Federici, 2005; Lecadet, 1996; Sanahuja
0022-2011/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jip.2012.05.001
Corresponding author at: Groupe dtude des protines membranaires, Univer-

sit de Montral, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, Canada H3C
3J7. Fax: +1 514 343 7146.
E-mail address: jean-louis.schwartz@umontreal.ca (J.-L. Schwartz).
Journal of Invertebrate Pathology 111 (2012) 112
Contents lists available at SciVerse ScienceDirect
Journal of Invertebrate Pathology
j our nal homepage: www. el sevi er. com/ l ocat e/ j i p
et al., 2011; Sanchis, 2011), in agriculture (Navon, 2000; Sanchis
and Bourguet, 2008; Sauka and Benintende, 2008) and forestry
(van Frankenhuyzen, 2000), and disease vectors (Becker, 2000; La-
cey and Undeen, 1986; Thiry et al., 1996). For the last decade and
a half, various transgenic crops that express insecticidal Bt toxins
have been grown over a rapidly increasing area (Cannon, 2000;
Ferr et al., 2008; James, 2010; Shelton et al., 2002).
Collectively, Bt strains produce a rather diverse group of toxin
proteins (de Maagd et al., 2003). In addition to those found in
the parasporal inclusions, insecticidal toxins designated Vip (vege-
tative insecticidal proteins) are secreted into the medium during
the vegetative phase of growth (Estruch et al., 1996). Most d-endo-
toxins belong to the Cry (crystal) family of proteins, but they also
include members of the Cyt (cytolytic) family, a group of proteins
found in diptericidal strains of Bt (Crickmore et al., 1998). The pro-
teins from these two families are structurally unrelated to each
other, but Cyt proteins interact with Cry proteins to potentiate
their effects, in a synergistic manner, against certain species of
mosquitoes and black ies (Butko, 2003; Federici et al., 2010;
Sobern et al., 2007a). Although the most extensively studied
members of the Cry family are insecticidal, nematocidal toxins
(Wei et al., 2003) and other Cry proteins known as parasporins
which are specically active against certain cancer cells (Ohba
et al., 2009) are presently receiving considerable attention. This re-
view, however, will focus on insecticidal Cry toxins with only occa-
sional reference to relevant information concerning other Bt toxins.
Because of their importance and long use in biological pest
management programs, considerable work has been devoted to
the elucidation of the mode of action of insecticidal Cry proteins
(Aronson and Shai, 2001; Chattopadhyay et al., 2004; Cooper,
1994; Dean et al., 1996; English and Slatin, 1992; Federici et al.,
2010; Gill et al., 1992; Himeno, 1987; Hone and Visser, 1993; Jur-
at-Fuentes and Adang, 2006a; Knowles, 1994; Knowles and Dow,
1993; Rajamohan et al., 1998; Schnepf et al., 1998; Sobern
et al., 2010; Whalon and Wingerd, 2003). By the end of the last
century, the sequence of events leading to insect death following
ingestion of Cry toxins appeared relatively simple and well under-
stood, at least in broad general terms (Rajamohan et al., 1998;
Schnepf et al., 1998). Crystal proteins are rst ingested as protoxins
which are solubilized and proteolytically converted to smaller,
protease-stable polypeptides, in the insect midgut. These activated
toxins then bind to specic receptors at the surface of midgut
epithelial cells, allowing them to insert into the membrane and
form poorly selective pores which are permeable to small
molecules such as inorganic ions, amino acids and sugars (Carroll
and Ellar, 1993; Kirouac et al., 2002). The presence of such pores
in the plasma membrane interferes with cell physiology by
abolishing transmembrane ionic gradients and may lead to
colloid-osmotic lysis of the cells due to the massive inux of
solutes from the midgut lumen (Knowles and Ellar, 1987). In turn,
destruction of the cells results in extensive damage to the midgut
epithelial tissue and death of the intoxicated larvae.
Many details of this scheme, which may be considered as the
classical model of Bt mode of action (Fig. 1), nevertheless remain
unresolved. For instance, the structure of the pores formed by the
toxins and the mechanism by which they assemble into the
Fig. 1. Schematic representation of the steps leading to pore formation and insect
death according to the classical model of Bt mode of action as summarized in
Section 1.
Fig. 2. Schematic representation of the steps leading to pore formation and insect
death according to the sequential binding model discussed in Section 2.
Fig. 3. Schematic representation of the steps leading to insect death according to
the signaling pathway model discussed in Section 3.
2 V. Vachon et al. / Journal of Invertebrate Pathology 111 (2012) 112
membrane have not been fully elucidated. On the other hand, con-
siderable progress has been accomplished in the identication of
Cry toxin receptors and understanding of their role in toxin recog-
nition as well as in resistance to these toxins (see Gmez et al.
(2007), Likitvivatanavong et al. (2011) and Pigott and Ellar
(2007) for detailed reviews). Otherwise, research on the mode of
action of Cry toxins has been dominated in recent years by two
models, the sequential binding model (Fig. 2), which proposes a
somewhat complex sequence of events involving multiple recep-
tors in an attempt to explain the mechanism of pore formation
(Bravo et al., 2004; Gmez et al., 2002; Pacheco et al., 2009a),
and the signaling pathway model (Fig. 3), which radically
questions the sequence of events presented above by suggesting
that pore formation does not play an essential role (Zhang et al.,
2005, 2006). A combination of the two above models has also been
proposed (Jurat-Fuentes and Adang, 2006a). The objective of the
present review is therefore to examine these two models in detail
and to critically evaluate the evidence on which they are based.
The review stresses the importance of a judicious use of experi-
mental techniques and careful interpretation of the resulting data
and underscores the need for much further effort in elucidating the
details of the mode of action of Bt Cry toxins.
2. The sequential binding model
This model, which is summarized in Fig. 2, has been extensively
reviewed by its authors in the last few years (Bravo et al., 2007,
2011; Bravo and Sobern, 2008; Jimnez-Jurez et al., 2008; Par-
do-Lpez et al., 2009, in press; Sobern et al., 2007a, 2009, 2010).
It describes a hypothetical mechanism of pore formation which is
largely based on work dealing with Cry1Ab and Manduca sexta. Like
the closely related toxins Cry1Aa and Cry1Ac, Cry1Ab is recog-
nized, in several lepidopteran insect species such as M. sexta, by
at least two specic receptors in the luminal membrane of midgut
epithelial cells: a cadherin-like protein and a glycosyl-phosphati-
dylinositol (GPI)-anchored aminopeptidase N (Gmez et al.,
2007; Likitvivatanavong et al., 2011; Pigott and Ellar, 2007).
According to the initial version of the sequential binding model,
once activated by intestinal proteases, the toxin binds to the cad-
herin. This causes a conformational change that favors a proteo-
lytic cleavage at the level of residue F50, located within the loop
linking helices a1 and a2, the rst helices on the N-terminal end
of the pore-forming domain of the toxin molecule (Gmez et al.,
2002). Subsequently, removal of helix a1 allows the rest of the tox-
in to oligomerize and form a so-called pre-pore structure (Gmez
et al., 2002). This oligomer then binds to the aminopeptidase
receptor as it possesses a much greater afnity for this protein than
the monomeric toxin (Bravo et al., 2004). Finally, binding to the
aminopeptidase favors the insertion of the pre-pore structure into
the membrane which is made more permeable by the resulting
pore. In a recent modication of this model (Pacheco et al.,
2009a), an additional step was proposed in which the monomeric
toxin rst binds to the aminopeptidase, with low afnity but high
capacity, before interacting with the cadherin, which is present in
smaller amounts in the membrane, as was pointed out earlier by
Pigott and Ellar (2007), but binds the toxin with higher afnity.
The main advantage of the sequential binding model resides in
the fact that it provides a conceptual framework for the experi-
mental study of the mechanism by which Bt Cry toxins form pores,
with each of its steps being, at least in principle, amenable to
experimental verication. In the following sections, we review crit-
ically the evidence supporting this model, the available data that
challenge it, and whether the additional steps it proposes, relative
to the classical activation ?binding ?pore formation ?cell lysis
scheme, are necessary for toxin activity.
2.1. Are two different receptors required?
The sequential binding model is particularly attractive in that it
tentatively explains why alterations in the binding properties or
the level of expression of either cadherins (Gahan et al., 2001;
Morin et al., 2003; Xu et al., 2005) or aminopeptidases N (Zhang
et al., 2009) can alone result in high levels of resistance to Bt toxins
even if some cases of resistance do not appear to be attributable to
modications in either one of these receptors (Baxter et al., 2005,
2008; Gahan et al., 2010; Higuchi et al., 2007; Khajuria et al.,
2011). Similarly, RNA silencing of either the cadherin (Fabrick
et al., 2009; Sobern et al., 2007b) or the aminopeptidase
(Rajagopal et al., 2002; Sivakumar et al., 2007) genes alone can
cause resistance to Cry toxins. The model is more difcult to recon-
cile, however, with the observation that heterologous expression of
either the cadherin receptors (Aimanova et al., 2006; Dorsch et al.,
2002; Flannagan et al., 2005; Hua et al., 2004a, 2004b; Jurat-Fuen-
tes and Adang, 2006b; Nagamatsu et al., 1999, 1998; Tsuda et al.,
2003; Zhang et al., 2005) or the aminopeptidase receptors
(Gill and Ellar, 2002; Sivakumar et al., 2007) alone can render the
host cells sensitive to one or several of the Cry1A toxins. In these
cases, according to the model, the untreated host cells would have
to possess the other type of receptor while being tolerant to the
toxins. This appears unlikely, however, since in most of these
studies, the toxins were shown to bind to the transformed cells,
but not to the control host cells. As mentioned above, these toxins
can bind to both receptors, albeit with different afnities (Bravo
et al., 2004; Pacheco et al., 2009a). Furthermore, the sequential
binding model is also difcult to reconcile with the results of
experiments demonstrating a large increase of
86
Rb
+
efux from
phospholipid vesicles enriched with partially puried aminopepti-
dase receptors (Luo et al., 1997; Sangadala et al., 1994), or the
formation of ion channels in planar lipid bilayer membranes into
which the puried aminopeptidase was incorporated, at a much
lower toxin concentration than in receptor-free membranes
(Schwartz et al., 1997b).
2.2. How critical is the removal of helix a1?
Proteolytic removal of helix a1 was demonstrated in studies in
which the Cry1Ab protoxin activation was performed in three dif-
ferent ways: (i) with trypsin and in the presence of scFv73, a sin-
gle-chain antibody which shares homology with a Cry1Ab-
binding region of the cadherin Cry1A toxin receptors of M. sexta
and Bombyx mori (Gmez et al., 2001); (ii) with M. sexta larval mid-
gut juice and in the presence of scFv73 (Gmez et al., 2001); or (iii)
in the presence of midgut brush border membrane vesicles puried
from M. sexta, which possess appropriate proteases and cadherin
receptors (Gmez et al., 2002). Following incubation and subse-
quent centrifugation of the samples, the activated toxin was recov-
ered in the soluble fraction as either 60-kDa monomers or what
appeared to be 120- and 250-kDa oligomers. The N-terminal se-
quence of the monomeric form of the toxin corresponded to the
N-terminus of helix a1, and that of the 250-kDa protein corre-
sponded to a segment starting at residue V51 and located between
helices a1 and a2. Unfortunately, the authors did not specify by
which of the three activation methods mentioned above the se-
quenced proteins were prepared or whether the same sequence
was obtained with each of these methods. In any case, it would ap-
pear surprising that trypsin could cleave between residues F50 and
V51, which is not a cleavage site for trypsin, but for chymotrypsin.
Cleavage of helix a1 following incubation of Cry1Ac with M. sex-
ta midgut brush border membrane vesicles had in fact been ob-
served earlier (Aronson et al., 1999). In that study, however, helix
a1 was only removed after the vesicles incubated with the toxin
were subsequently treated with proteinase K. Although the study
V. Vachon et al. / Journal of Invertebrate Pathology 111 (2012) 112 3
of Aronson et al. (1999) is often cited in support of the sequential
binding model, the activated toxin clearly remained intact in the
vesicles that were not exposed to the added protease.
The necessity of cleaving off helix a1 for toxin activity, which
constitutes one of the critical features of the sequential binding
model, is also questionable for other reasons. Firstly, it has been
observed that the rate of pore formation by Cry1Aa in M. sexta mid-
gut brush border membrane vesicles was not inuenced by a wide
variety of protease inhibitors, including several inhibitors of serine
proteases which constitute the main protease type in the midgut of
lepidopteran insects (Kirouac et al., 2006b). Furthermore, when the
cleavage site which is thought to be involved in the removal of he-
lix a1 (Gmez et al., 2002) was eliminated in the Cry1Aa mutant
F50C, permeabilization of the midgut vesicles was signicantly
slower, as with several other domain I mutants, than with the
wild-type Cry1Aa toxin (Lebel et al., 2009). Nevertheless, in the
presence of the F50C mutant, pore formation did occur in M. sexta
midgut brush border membrane vesicles and their permeability
was almost identical following a 1-h preincubation of the vesicles
with either the mutant or the parental toxins (Lebel et al., 2009).
Actually, further proteolysis of the activated Cry1Ab toxin, in
the presence of the midgut epithelial cell membrane, appears to
hinder its activity rather than being necessary for pore formation
and toxicity, as demonstrated by micro-electrode measurements
of the intracellular electrical potential of the epithelial cells from
freshly isolated M. sexta midguts (Fortier et al., 2007a). Whereas
the rate at which Cry1Ab reduced the intracellular potential in-
creased signicantly in the presence of M. sexta midgut juice, this
effect was also observed either in the presence of boiled midgut
juice, or with a cocktail of protease inhibitors in the absence of
midgut juice (Fortier et al., 2007a).
Strong support in favor of the helix a1 removal step of the
sequential binding model has been suggested by experiments con-
ducted with Cry1AbMod and Cry1AcMod, two genetically modied
Cry1Ab and Cry1Ac proteins that lack the 56 N-terminal amino
acids of the protoxin, and therefore all of helix a1, and have two
amino acid substitutions in helix a2 (Sobern et al., 2007b). These
modied toxins were found to be toxic to M. sexta and Pectinophora
gossypiella larvae that were resistant to the corresponding unmod-
ied toxins (Sobern et al., 2007b) due, respectively, to either a re-
duced level (Sobern et al., 2007b) or the absence (Morin et al.,
2003) of functional cadherin receptors. They were also toxic to
greenhouse-selected populations of Trichoplusia ni that were resis-
tant to Cry1Ab and Cry1Ac (Franklin et al., 2009). Although the
sequential binding model offers an appealing explanation for these
remarkable results, they do not prove that the removal of helix a1
is actually necessary for toxin activity. Indeed, if the modied tox-
ins lacking helix a1 simply bypassed the cadherin binding step,
both the modied and unmodied toxins would be expected to
have similar toxicities towards susceptible insect strains. In fact,
when tested against a susceptible strain of P. gossypiella, Cry1Ab-
Mod was 3.5 times less toxic than Cry1Ab, and Cry1AcMod was
86 times less toxic than Cry1Ac (Sobern et al., 2007b). Similarly,
Cry1AbMod was about twofold less toxic than Cry1Ab and Cry1Ac-
Mod, 14.5-fold less toxic than Cry1Ac, when tested against suscep-
tible Trichoplusia ni larvae (Franklin et al., 2009). In the case of
Cry1AbMod, these differences are admittedly small, but they ap-
pear quite signicant in view of the 95% ducial limits reported
in these experiments. It should be mentioned, however, that
Cry1Ab and Cry1AbMod were since reported to be equally toxic
to toxin-sensitive M. sexta larvae in a more recent study (Muoz-
Garay et al., 2009a). Nevertheless, a recent study (Tabashnik
et al., 2011) provided a rather convincing demonstration that the
difference between the activity of these modied toxins and those
of their respective wild-type parental toxins is not simply attribut-
able to the possibility that the modied toxins, because they lack
helix a1, may function without the need to interact with the cad-
herin receptor. In this study, Cry1AbMod and Cry1AcMod were
tested against several insect strains resistant to Cry1Ab and Cry1Ac
and found to have enhanced activity, relative to the wild-type tox-
ins, against some insects in which resistance is not related to a
mutation affecting the cadherin receptor. Furthermore, modied
and wild-type toxins had comparable potencies against other resis-
tant insect strains with altered cadherin.
In summary, although removal of helix a1 from activated Cry
toxins may occur and possibly improve toxin activity under certain
circumstances, the evidence presently available does not support
the idea that such cleavage constitutes a necessary step in Bt toxin
mode of action.
2.3. Is a toxin pre-pore required for membrane insertion?
Fairly early on, several authors have proposed that Bt toxin
pores were oligomeric structures within the membrane (Gazit
and Shai, 1995; Hodgman and Ellar, 1990; Schwartz et al.,
1997a). More recently, several research groups have devoted con-
siderable effort to identify such oligomers and to characterize their
structural and functional properties (Aronson et al., 1999; Gmez
et al., 2002; Ihara and Himeno, 2008; Kumar and Aronson, 1999;
Likitvivatanavong et al., 2006; Obata et al., 2009; Tigue et al.,
2001; Xiang et al., 2009). Although some of these studies are regu-
larly cited in support of the sequential binding model, the tech-
niques used to obtain oligomers are often very different and so
are, in many cases, the biochemical properties of the resulting
proteins.
Most studies on the basis of which the sequential binding mod-
el has been elaborated describe oligomers that were obtained by
activating the Cry1Ab protoxin, during an hour or less, in the pres-
ence of cadherin or peptides corresponding to different regions of
this receptor (Gmez et al., 2002; Pacheco et al., 2009b). Similar
oligomers were also observed by incubating the Cry3Aa protoxin
with Leptinotarsa decemlineata (Rausell et al., 2004a) or Tenebrio
molitor (Fabrick et al., 2009) brush border membrane vesicles, or
the Cry1Ca protoxin with vesicles isolated from Spodoptera exigua
(Herrero et al., 2004). Oligomers were also observed when the Bt
var. israelensis Cry11Aa protoxin was activated with trypsin in
the presence of vesicles from Aedes aegypti (Prez et al., 2007).
The formation of Cry11Aa oligomers was signicantly promoted
by Cyt1Aa, suggesting that, similar to the role played by cadherins
in the sequential binding model for Cry1 and Cry3 toxins, Cyt1Aa
serves as an oligomerization-promoting receptor for Cry11Aa
(Muoz-Garay et al., 2009b; Prez et al., 2005, 2007).
As mentioned before, activation of Cry1A protoxins in the pres-
ence of their receptors resulted in the formation of toxin aggre-
gates of 120 and 250 kDa (Gmez et al., 2001, 2002).
Surprisingly, however, these structures were not visible upon so-
dium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
and were apparently only detected, in rather small amounts com-
pared with the monomers, by Western blotting using anti-Cry1Ab
polyclonal antibodies (Gmez et al., 2006, 2002). Under the condi-
tions used, activation of the protoxin into monomeric toxin thus
appears to be very much more efcient than oligomerization. In
addition, the 250-kDa structures, like the oligomers described by
Zhang et al. (2005), were shown to be highly stable since they were
not disrupted by boiling in the presence of SDS (Gmez et al.,
2002). This is in sharp contrast with the properties of the oligomers
described in earlier reports (Aronson et al., 1999; Tigue et al.,
2001). In these studies, M. sexta brush border membrane vesicles
were incubated with trypsin-activated Cry1Ab or Cry1Ac, washed
extensively, and extracted with SDS at 70 C (Tigue et al., 2001)
or with the milder detergent octyl-b-D-glucopyranoside at 65 C
(Aronson et al., 1999) before Western blot analysis. The major
toxin species detected in these membrane extracts had molecular
masses of 120130 kDa and 190200 kDa (Aronson et al., 1999;
Kumar and Aronson, 1999), or 65, 130 and 200 kDa (Tigue et al.,
2001). When the samples were boiled before electrophoresis, how-
ever, only the monomeric form of the toxin was detected (Aronson
et al., 1999).
Temperature sensitivity, in the presence of SDS, does not appear
to be related to the use of activated toxins since oligomers of
250 kDa prepared by activating the Cry1Ac protoxin with trypsin
and Helicoverpa armigera midgut brush border membrane vesicles
were also readily detected after incubation at 60 C, but not after
boiling in electrophoresis loading buffer (Xiang et al., 2009). Oligo-
merization of proteolytically-activated and
125
I-labeled Cry1Aa
was assayed in the presence of either B. mori midgut brush border
membrane vesicles or proteins solubilized from such vesicles with
cholic acid (Ihara and Himeno, 2008). After incubation with the la-
beled toxin, the vesicles were further exposed to unlabeled Cry1Aa
and nally centrifuged before analysis by autoradiography to en-
sure that the analyzed toxins were irreversibly bound. Only mono-
mers were detected in the samples obtained with solubilized
membrane proteins, but 250-kDa oligomers were produced in
the presence of the vesicles. These oligomers were readily detected
in samples incubated at 40 or 60 C before electrophoresis, but dis-
appeared when the samples were heated to 80 or 100 C. Unfortu-
nately, the authors did not verify whether the cadherin receptors
were actually solubilized from the vesicles, but the likelihood that
they were indicates that the presence of cadherins is obviously not
sufcient for oligomerization to occur. On the other hand, the pres-
ence of an appropriate membrane appears essential. In agreement
with this suggestion, temperature-sensitive trimers of Cry4Ba were
obtained by incubating the trypsin-activated form of this toxin
with liposomes composed of a mixture of phosphatidylcholine,
phosphatidylethanolamine and cholesterol (Likitvivatanavong
et al., 2006).
More recently, oligomerization was assayed by Western blot
analysis after incubating trypsin-activated Cry1Aa with B. mori
midgut brush border membrane vesicles (Obata et al., 2009). This
yielded a toxin species that ran well above the band formed by
the 200-kDa standard. The amount of this oligomeric structure that
was produced increased considerably between 15 min and 2 h of
incubation with the vesicles, but disappeared almost completely
when the samples were heated at 70 or 100 C before the electro-
phoresis step. The larger than 200-kDa toxin oligomer was also
produced in similar quantities by incubating the activated toxin
with a 27-kDa fragment of the B. mori cadherin comprising its tox-
in-binding region, in the absence of added protease (Obata et al.,
2009). Unfortunately, the authors did not report the N-terminal
amino acid sequence of the components of the oligomer produced
under these conditions, but its formation in the absence of prote-
ases suggests that further proteolysis of the activated toxin is not
necessary for oligomerization. For one of the mutants studied,
however, proteolysis did occur within the loop between helices
a2a and a2b during incubation with the vesicles (Obata et al.,
2009). This mutant, S373C, as well as several other single-site mu-
tants, was able to oligomerize spontaneously during activation in
the absence of cadherin. Oligomerization of activated toxin is
therefore not restricted to the engineered Cry1AbMod and Cry1Ac-
Mod toxins lacking helix a1 (Sobern et al., 2007b), as discussed at
the end of Section 2.2.
Further analysis of the Cry1AbMod toxin has indicated that its
oligomerization during activation in the presence of trypsin, but
in the absence of cadherin, is highly dependent on pH (Muoz-
Garay et al., 2009a). Unfortunately, the authors did not specify
the source of the trypsin they used for these experiments. Never-
theless, as toxin activation with trypsin is usually done with
commercial preparations of either bovine or porcine origin, it
appears surprising that the amount of oligomers produced by incu-
bating the toxin with trypsin, in the absence of membranes, be-
came signicant only at pH values above 10 at which trypsins of
mammalian origin are unlikely to function efciently.
The biochemical characteristics of the oligomeric structures
supposedly forming pre-pores and prepared by activating protox-
ins in the presence of cadherin appear difcult to reconcile with
those of the toxin proteins extracted from membranes after incu-
bation with the activated toxins. In fact, little attention has been
devoted to the physical state of these pre-pores once inserted in
the membrane (Pardo-Lpez et al., 2006). In one study, however,
Cry1Ab was incubated with M. sexta brush border membrane
vesicles, and the unbound toxin was removed by centrifugation
and extensive washing of the vesicles before Western blot analysis
using anti-Cry1Ab antibodies (Pacheco et al., 2009a). Very surpris-
ingly, only monomers were reported (Pacheco et al., 2009a), in
complete contradiction with the sequential binding model accord-
ing to which the toxin must oligomerize before inserting into the
membrane and forming pores (Bravo et al., 2004). In similar exper-
iments, previously trypsin-activated and biotinylated Cry1Aa,
Cry1Ab and Cry1Ac were recovered, apparently only in their mono-
meric form, from brush border membrane vesicles, following
extensive washing of the vesicles after incubation with the toxins
(Bravo et al., 2002; Gmez et al., 2001; Padilla et al., 2006). Accord-
ing to the model, at least a substantial proportion of the toxin
molecules should have been found as oligomers, unless biotinyla-
tion prevented their oligomerization or insertion into the mem-
brane. This appears unlikely, however, since it was mentioned
that the toxicity of the biotinylated toxin is similar to that of the
unlabeled toxin (Padilla et al., 2006). It is thus difcult to imagine
how the very highly stable structures formed by the so-called pre-
pores would become fragile to the point of dissociating into mono-
mers after insertion into the membrane. However, because the
samples were boiled before electrophoresis, at least when specied
by the authors (Bravo et al., 2002; Pacheco et al., 2009a; Padilla
et al., 2006), the detection of all the membrane-associated toxin
in the form of monomers, although in contradiction with the
model, is actually consistent with the observations of practically
all the other research groups whose data were discussed above.
More evidence that the physical characteristics of the toxin olig-
omers depend on the method with which they are prepared can be
obtained by comparing the results of studies designed to demon-
strate the association of toxin molecules with lipid rafts (Bravo
et al., 2004; Zhuang et al., 2002). Because GPI-anchored membrane
proteins, including aminopeptidase receptors, are found within
these detergent-resistant membrane lipid subdomains, most of
the membrane-bound toxin indeed co-puries with these struc-
tures, at least when the experiments are carried out in the presence
of relatively lowconcentrations of toxin (Zhuang et al., 2002). When
lipid rafts were isolated from M. sexta or Heliothis virescens midgut
brush border membrane vesicles that had been previously incu-
bated with activated and biotinylated Cry1Ac, most of the toxin
was found associated with this lipid fraction as a single band which
presumably corresponds to its monomeric form since the authors
make no mention of its apparent molecular mass or of the presence
of oligomers (Zhuang et al., 2002). This result is in sharp contrast
with that obtained when lipid rafts were isolated fromM. sexta mid-
gut brush border membrane vesicles that had been previously incu-
batedwiththeCry1Abprotoxin(Bravoet al., 2004). Inthis case, most
of the toxinwas detected, associatedwithlipidrafts, as a widespread
streak of protein ranging in apparent molecular mass from well be-
low160 kDa to well above 250 kDa, which was interpreted as corre-
sponding to its oligomeric form (Bravo et al., 2004). Differences in
the properties of the toxin oligomers are nevertheless not always
attributable to the use of different techniques for their preparation.
In a recent study aimed at probing the structure of the toxin
molecule once inserted into the membrane (Zavala et al., 2011), a
number of activated Cry1Ab mutants were incubated individually
with M. sexta brush border membrane vesicles under conditions
which appear to be very similar to those used in the above-men-
tioned studies, at least as far as can be judged fromthe experimental
details provided by the authors. In contrast with the results dis-
cussed above, all tested mutants analyzed by Western blot using
an anti-Cry1Ab polyclonal antibody, with only one exception, were
detected mainly as 250-kDa structures, with in most cases practi-
callynodetectable65-kDa monomeric protein(Supplemental mate-
rial of Zavala et al. (2011)).
It has been suggested earlier that pore formation in Sf9 cultured
insect cells may result from the insertion into the membrane of a
pre-assembled multimeric toxin structure on the basis of a detailed
analysis of toxin-induced K
+
-efux from these Bt-sensitive cells de-
rived from the ovary of Spodoptera frugiperda larvae (Guihard et al.,
2000). However, in the case of the midgut of Bt-sensitive insects,
the need for pre-oligomerization is difcult to reconcile with the
results of experiments performed with nontoxic mutants that were
unable to form oligomers and to permeabilize M. sexta midgut
brush border membrane vesicles, but were unaffected in their abil-
ity to bind to such vesicles (Cooper et al., 1998; Tigue et al., 2001).
The observation that these mutants bound irreversibly to the ves-
icles, a process which is considered to correspond mostly to the
insertion of the toxin into the membrane (Ihara and Himeno,
2008; Liang et al., 1995), strongly suggests that oligomerization
follows membrane insertion (Tigue et al., 2001). This sequence of
events is clearly in contrast with the predictions of the sequential
binding model (Bravo et al., 2004). Further evidence of the possibil-
ity that Cry toxins can insert into the membrane as monomers was
obtained in a recently published single molecule uorescence
study involving tetramethylrhodamine-labeled Cry1Aa toxin
inserted into supported lipid bilayers (Groulx et al., 2011). Fluores-
cence photobleaching analysis revealed the presence of toxin
monomers, dimers, trimers and tetramers in these membranes
prepared from phospholipid liposomes pre-incubated with the
trypsin-activated and uorescently labeled toxin.
In summary, those highly stable structures supposedly constitut-
ing pre-pores do not appear to be easily detected, as they should
according to the sequential binding model, in target membranes ex-
posedtothe toxin. Onthe other hand, toxinmonomers are readilyex-
tracted from such membranes. These results indicate that toxin
oligomers couldpossiblyassemblewithinthe membrane frommem-
brane-inserted toxin monomers. They are also compatible, however,
with the possibility that toxin oligomers could be easily destabilized
by the presence of detergents and the experimental conditions used
for their solubilization and analysis which usually involves an elec-
trophoresis step. The possibility therefore remains that pre-pores
could form at the surface of the membrane even though such struc-
tures may be difcult to isolate from the membrane without being
disrupted. Innovative experimental approaches will undoubtedly
be required to distinguish between these possibilities.
2.4. Are pre-formed oligomers more efcient than toxin monomers?
Within the context of the sequential binding model, consider-
able effort has been devoted to demonstrate that the monomeric
form of the toxin has only a very poor pore-forming ability (Gmez
et al., 2002; Muoz-Garay et al., 2006; Pardo-Lpez et al., 2006;
Prez et al., 2007; Rausell et al., 2004a,b,c). This conclusion is very
surprising in that it contradicts decades of work in this eld. To cite
only a few examples, in vitro trypsin-activated monomeric toxins
do efciently cause structural damage to the midgut epithelium
(Bravo et al., 1992), permeabilize the plasma membrane of
sensitive cultured insect cells (Guihard et al., 2000; Knowles and
Ellar, 1987; Schwartz et al., 1991; Vachon et al., 1995; Villalon
et al., 1998), abolish the membrane potential of freshly isolated in-
sect midguts (Peyronnet et al., 1997), inhibit short-circuit currents
generated across the isolated insect midgut epithelium (Chen et al.,
1993; Liebig et al., 1995), permeabilize insect midgut brush border
membrane vesicles (Carroll and Ellar, 1993; Coux et al., 2001; Kir-
ouac et al., 2006a; Tigue et al., 2001) and inhibit amino acid trans-
port into these vesicles (Sacchi et al., 1986; Wolfersberger, 1991).
Moreover, most experiments comparing the permeabilization abil-
ity of pre-formed oligomers with that of monomers (Gmez et al.,
2002; Muoz-Garay et al., 2006; Pardo-Lpez et al., 2006; Prez
et al., 2007; Rausell et al., 2004a,b,c) were done under conditions
that allowed the monomeric toxins to pass through every step of
the mechanism of pore formation, including possibly those de-
scribed by the sequential model. In such experiments, given suf-
cient time for the toxin to insert into the membrane, whether in
the form of pre-formed oligomers or monomers, and the mono-
mers to oligomerize, both monomers and oligomers should be
equally active. It is therefore difcult to understand why Cry1Ab
activated with M. sexta midgut juice was, as reported, 20.7 times
less toxic than the Cry1Ab protoxin and 12.9 times less toxic than
the Cry1Ab protoxin activated with scFV73 and midgut juice
(Gmez et al., 2002). Also difcult to reconcile with the model is
the observation that a dose of Cry1Ab oligomers vefold lower
than the 50% lethal concentration measured for the trypsin-
activated Cry1Ab caused 95% mortality among M. sexta neonate
larvae after 5 days (Pacheco et al., 2009a).
Comparing the activity of pre-formed oligomers and monomers
may nevertheless provide a useful test for the sequential binding
model, but such experiments must take into account the kinetics
of pore formation. The outcome of such experiments will depend
on whether the oligomerization of monomers or the insertion of
Fig. 4. Representative channel current traces recorded at two opposite holding
voltages across a phosphatidylethanolamine-phosphatidylcholine-cholesterol
(7:3:2, w/w) planar lipid bilayer into which trypsin-activated Cry1Ab (80 nM)
was inserted under symmetrical 150 mM KCl conditions, pH 9.0. Upwards jumps at
+40 mV and downwards jumps at 40 mV correspond to the current owing
through the channels in their open state. Dotted lines (marked C) indicate the
zero current, corresponding to all channels being in their closed state. Records
display large jumps corresponding to a channel conductance of 580 pS (from three
similar experiments), and a smaller transition of about 200 pS that may represent a
subconducting state of the channels.
the toxinconstitutes the rate-limiting stepinthe mechanismof pore
formation. According to the model, toxin monomers are expected to
have a lower pore-forming ability than oligomers when the experi-
mental conditions prevent, or at least fail to favor, their oligomeriza-
tion. This should be the case in experiments carried out with
receptor-free lipid bilayer membranes (Pardo-Lpez et al., 2006;
Rausell et al., 2004b). In agreement with the suggested involvement
of pre-pores in the mechanism of action of Cry toxins, the mono-
meric formof Cry1Ab was reported to cause only noisy uctuations
in the planar lipid bilayers, whereas well-resolved channel currents
were observed with puried oligomers at a concentration at least
20-fold lower than that used for the Cry1Ab monomers (Pardo-
Lpez et al., 2006; Rausell et al., 2004b). The absence of well-dened
current steps produced in planar lipid bilayers by the monomeric
toxin is very surprising, however, in light of the fact that single-
channel currents induced by a variety of other activated Cry toxins,
including Cry1Aa (Grochulski et al., 1995), Cry1Ac (Slatin et al.,
1990), Cry1Ca (Lorence et al., 1995; Schwartz et al., 1993), Cry2Aa
(English et al., 1994), Cry3Aa (Slatin et al., 1990), Cry4Ba (Puntheer-
anurak et al., 2004), and the Cry34Ab/Cry35Ab binary toxin (Masson
et al., 2004), have been described. Actually, as shown in Fig. 4,
Cry1Ab does not constitute an exception, and single-channel con-
ductance steps are readily demonstrated withthe trypsin-activated,
and therefore monomeric, form of this toxin.
Comparisons between the activity of monomers and oligomers
were also made with a membrane potential-sensitive uorescent
dye, diS-C
3
(5) (Gmez et al., 2002; Muoz-Garay et al., 2006), using
a technique which has been extensively used to study Cry toxins.
This approach represents a powerful tool for the study of pore for-
mation by Bt toxins. It nevertheless requires the use of appropriate
experimental protocols to avoid misinterpretation of rather easily
acquired data, as was critically assessed in detail by Kirouac et al.
(2003). Unfortunately, however, the main arguments and warnings
put forward by these authors appear to have been completely ig-
nored by the other users of that uorescence technique (Gmez
et al., 2002; Gonzlez-Cabrera et al., 2006; Leonardi et al., 2007;
Muoz-Garay et al., 2006; Padilla et al., 2006; Rausell et al.,
2004a,c). As discussed earlier in more detail (Kirouac et al., 2003),
because the pores formed by Cry1Ab, and many other Cry toxins,
are not strongly cation-selective, the uorescence level changes
caused by the toxin are very small. This is illustrated by the fact that
valinomycin had a much stronger effect than Cry1Ab oligomers on
the measured uorescence levels, even at very low ionophore con-
centration (0.1 lM) (Muoz-Garay et al., 2006). Contrary to what
was claimed in that study, a high concentration of valinomycin will
not abolish the ionic gradients in this type of experiment, but rather
ensure that the largest possible potential is generated across the
membrane. On the other hand, in several reports (Padilla et al.,
2006; Rausell et al., 2004a,c), it was shown that valinomycin itself
hadverylittle effect onthe measureduorescence levels, suggesting
that the vesicle preparations containedonlya fewtightlysealedves-
icles. It cannot be excluded, however, that the small effect observed
in these studies was due to the addition of a suboptimal amount of
the ionophore, whose dose was not specied by the authors. In
any case, the toxins tested this way erroneously appeared to have
an activity that compared to that of valinomycin. Therefore, the
above-mentioned membrane potential measurement studies do
not provide convincing evidence that toxin oligomers are more ac-
tive than monomers, because the recorded differences in uores-
cence levels depended mainly on the ionic selectivity of the toxin
rather than on its ability to form pores. In addition, the effect of
the duration of vesicle exposure to the toxins could not be evaluated
from the experiments, as performed.
In principle, among the techniques used until now to compare
the activity of monomers and oligomers, the calcein release assay
is by far the most reliable for evaluating the rate at which a pore-
forming toxin permeabilizes its target membrane. In this technique,
calcein-loaded vesicles are incubated with the toxin. As the toxin
permeabilizes the membrane, calcein is released from the vesicles
and its uorescence increases due to dequenching (Kayalar and
Dzgnes, 1986). The experiments performed using this approach
didnot reveal a different rate of membrane permeabilizationby tox-
inmonomers andoligomers (Prez et al., 2007; Rausell et al., 2004a).
Instead, althougholigomers were shownto cause a muchgreater re-
lease of calcein than trypsin-activated toxin monomers, both forms
of the toxin appeared to act instantaneously, at least withinthe time
resolution of the instrument, upon addition of the toxin to the vesi-
cle suspension (Prez et al., 2007; Rausell et al., 2004a). This result is
verysurprising considering that althoughcalceinrelease canbe very
rapid, it is nevertheless expected to take place at a measurable rate
(Schwarz and Robert, 1990) as was reported for the Escherichia coli
colicin E1 (Kayalar and Dzgnes, 1986) and hemolysin (Menestri-
na, 1988), the Clostridium tetani tetanus toxin (Menestrina et al.,
1989), the Pseudomonas aeruginosa exotoxin A (Menestrina et al.,
1991) and the mosquitocidal binary toxin from Bacillus sphaericus
(Schwartz et al., 2001), tomentiononlya fewexamples of calcein-ef-
ux experiments conducted with bacterial pore-forming toxins.
Clearly, pore formation by Bt toxins is not expected to occur instan-
taneously, but requires some time for their insertion into the mem-
brane. For instance, the effect of Cry1Ac on the permeability of M.
sexta midgut brush border membrane vesicles only became evident
after a delay of about 10 s when measured under optimized condi-
tions (Fortier et al., 2007b). Similarly, Cry toxin-induced depolariza-
tion of the apical membrane of the epithelial cells of isolated
lepidoteran larval midguts (Fortier et al., 2007a; Peyronnet et al.,
1997), and inhibition of short-circuit currents across the larval mid-
gut epithelium (Chen et al., 1993; Liebig et al., 1995) could only be
observed after 1 min or more following exposure to the toxin.
Instantaneous changes in uorescence levels such as those men-
tioned above (Prez et al., 2007; Rausell et al., 2004a) are similar
to those that would be observed upon addition of a detergent, sug-
gesting that the vesicles used in these studies may have been dam-
aged independently of Cry toxin activity. Indeed, osmotic lysis of the
vesicles cannot take place in this type of experiment since, apart
fromcalcein itself, which is present at a higher concentrationwithin
the vesicles, and the toxin, whichis added to the extravesicular mili-
eu, the vesicles contain the same medium as that in which they are
suspended (Prez et al., 2007; Rausell et al., 2004a). More recently,
very different kinetics of calcein release were observed (Rausell
et al., 2007) in experiments performed with vesicles prepared from
the same insect species and the same toxin as earlier (Rausell et al.,
2004a), but unfortunately these new experiments did not directly
address the issue of monomer vs oligomer activity of Cry toxins.
In summary, efforts to demonstrate that pre-pore structures are
more active than monomeric toxins have not yielded convincing re-
sults. In part, this is probably attributable to the use of questionable
experimental protocols. However, as discussed in the previous sec-
tion, there is strong evidence that toxins can insert into the mem-
brane as monomers. It is therefore very likely that functional pores
assemble within the membrane after the insertion step. As was also
argued in the summary of Section 2.2, although it cannot be com-
pletely excluded at present that pore formation by Bt Cry toxins
could involve a pre-pore intermediate, it remains to be
demonstrated that the toxin aggregates tested so far as putative
pre-pore structures are actuallypart of the general mechanismof ac-
tion of Bt Cry toxins.
3. The signaling pathway model
According to this model (Fig. 3), cytotoxicity is mediated by the
specic binding of Bt toxins to their cadherin receptors. This acti-
vates otherwise undescribed Mg
2+
-dependent (Zhang et al., 2005)
and adenylyl cyclase/protein kinase A (Zhang et al., 2006) signaling
pathways that lead to necrotic cell death. While the toxins can
interact non-specically with membrane lipids, assemble into olig-
omers and even insert into the membrane, this has no consequence
for the target cells because, as the authors claim ((Zhang et al.,
2005), caption of Fig. 6), membrane-incorporated oligomer com-
plex does not form lytic pores in the membrane and has no toxic
effect on cells.
First and probably foremost among its problems, this model
simply ignores over two decades of work on Bt insecticidal Cry tox-
ins. As was discussed earlier in detail (Schwartz and Laprade,
2000), pore formation has been demonstrated unambiguously
and repeatedly using a variety of experimental approaches includ-
ing conductance measurements in planar lipid bilayer membranes,
permeability assays based on light scattering or uorescence mea-
surements performed on insect midgut brush border membrane
vesicles, on membrane potential or short-circuit current measure-
ments on isolated midguts, and on osmotic swelling experiments
on cultured insect cells.
The signaling pathway model is partially based on the observa-
tion that S5 cells, High Five (H5) cells derived from the cabbage
looper T. ni and transformed to express a cadherin receptor from
M. sexta, have acquired sensitivity to the Bt Cry1Ab toxin and that
ethylene diamine tetraacetic acid (EDTA), but not ethylene glycol
tetraacetic acid (EGTA), prevents cellular damage caused by the
toxin, as evaluated by Trypan blue staining (Zhang et al., 2005).
These divalent cation chelators differ in that EGTA binds magne-
sium less efciently than EDTA, at least at near neutral pH (Sch-
warzenbach and Flaschka, 1969). However, in M. sexta brush
border membrane vesicles, an experimental system in which intra-
cellular signaling pathways cannot be functioning because many of
their elements are missing, EDTA was also shown to strongly inhi-
bit pore formation by Cry1Aa, Cry1Ab, Cry1Ac and Cry1Ea, but not
Cry1Ca (Kirouac et al., 2006b). In these experiments, carried out in
the nominal absence of divalent cations, EGTA was also able to in-
hibit pore formation, but only at pH 10.5 at which its chelating
capacity, in particular for magnesium, is much greater than at neu-
tral pH (Schwarzenbach and Flaschka, 1969). The role of divalent
cations in toxin activity thus exerts itself, at least in part, at the le-
vel of pore formation (Fortier et al., 2005; Kirouac et al., 2006b).
Furthermore, the experiments presented by Zhang et al. (2005)
were apparently carried out in Insect-Xpress medium, but the
authors make no mention of the pH, which is between 6.2 and
6.4 (A. Ellis, Lonza Walkersville Inc., Walkersville, MD, USA, per-
sonal communication), and the divalent cation content of this
medium, which contains 7.6 mM MgSO
4
and 4.5 mM CaCl
2
(Ellis,
personal communication). The 5 mM EDTA or EGTA concentration
used by Zhang et al. (2005) was therefore insufcient to remove
completely the divalent cations from the medium, which raises
doubts on the Mg-dependence of their observations.
In support to their model, Zhang et al. (2006) presented an
experiment in which the effect of Cry1Ab on the morphology of
S5 cells was monitored in the presence of glucose, sucrose or raf-
nose. A close examination of their data shows, however, that the
cells clearly swelled in the presence of either one of these osmo-
protecting agents. Swelling was most obvious in the presence of
glucose. Cells treated with Cry1Ab in the presence of sucrose or
rafnose also swelled, but displayed the formation of blebs at the
cell surface. Blebbing was most clearly observed in the presence
of rafnose. Although Zhang et al. (2006) take these observations
as evidence that cell death occurs without membrane permeabili-
zation, the demonstration that these sugars inhibit osmotic swell-
ing and lysis of cultured insect cells in the presence of Bt toxins to
which they are sensitive was precisely one of the major arguments
originally put forward by Knowles and Ellar (1987) as evidence of
pore formation by the toxins. As glucose, a monosaccharide, is
smaller than sucrose, a disaccharide, and rafnose, a trisaccharide,
it is expected to have the least protecting effect since it can pene-
trate most rapidly into the cells through the pores formed by the
toxin. Conversely, rafnose being considerably larger will diffuse
much slower across the cell membrane and protect the cells more
efciently than the other two sugars.
In fact, the signaling pathway model even contradicts some of
the arguments the authors put forward in its support. The conclu-
sion that the toxin did not form pores was made from the observa-
tion of 140- and 240-kDa oligomers which were formed after
incubation of the monomeric toxin with either toxin-tolerant H5
cells, which do not express the cadherin receptor, or toxin-sensi-
tive S5 cells (Zhang et al., 2005). The authors nevertheless stated
(Zhang et al., 2005) that: Studies of mutated Cry toxin proteins
have shown that neither the toxin oligomer complex nor commen-
surate changes in membrane vesicle permeability correlate di-
rectly with toxicity, citing Kumar and Aronson (1999), Luo et al.
(1999) and Vachon et al. (2002, 2004). It is difcult to imagine,
to say the least, how the toxins can function without either form-
ing pores or increasing signicantly the activity of endogenous
membrane transporters and, at the same time, change membrane
permeability. In turn, increasing membrane permeability cannot
be as inconsequential as suggested (Zhang et al., 2005), since io-
nic gradients maintained across the plasma membrane, at a great
energetic expense for the cell (Harvey et al., 1983, 1998), are obvi-
ously crucial for cell viability.
In addition, the sentence cited in the previous paragraph
ignores the fact that toxicity is evaluated from the ability of the
toxins to kill insect larvae in which the midgut biochemical envi-
ronment is much more complex than that of the solutions bathing
brush border membrane vesicles in the in vitro experiments per-
formed to evaluate the toxins pore-forming ability (Kumar and
Aronson, 1999; Luo et al., 1999; Vachon et al., 2002, 2004). Further-
more, the discrepancy between in vitro and in vivo activities of the
mutants analyzed in these studies is far from being as obvious as it
is suggested by Zhang et al. (2005). For instance, Kumar and Aron-
son (1999) indeed describe mutants that were nontoxic but able to
oligomerize following incubation with M. sexta brush border mem-
brane vesicles. These mutants were nevertheless shown to be un-
able to permeabilize such vesicles in a light-scattering assay
(Kumar and Aronson, 1999). In the case of our own studies (Vachon
et al., 2002, 2004), the correlation between toxicity and pore-form-
ing ability was in fact rather good even though a few exceptions
were pointed out. Among these, the Cry1Aa mutant R128C, which
is toxic, but did not form pores efciently (Vachon et al., 2004), was
further studied and shown to form dimers that are stabilized by a
disulde bridge (Girard et al., 2009). When this bond was broken,
either by the reducing conditions of the midgut lumen or the addi-
tion of a reducing agent in the light-scattering assay, this mutant
was highly active, in agreement with its strong toxicity (Girard
et al., 2009). In fact, considerable work has been devoted to the
study of the inuence on Cry toxin activity of insect midgut bio-
chemical and biophysical factors, such as pH (Fortier et al., 2005;
Tran et al., 2001; Vachon et al., 2006), ionic strength (Fortier
et al., 2005), temperature (Vachon et al., 2006), presence of
divalent cations (Fortier et al., 2005; Kirouac et al., 2006b) and
proteases (Fortier et al., 2007a; Kirouac et al., 2006b), and reducing
conditions (Girard et al., 2009). These studies, which have been lar-
gely ignored so far, demonstrate clearly that critical consideration
of the role of such factors must be given to evaluate the differences
between in vivo and vitro data, and therefore to understand Bt tox-
in function.
Furthermore, the signaling pathway model simply ignores, on
the basis of no experimental data or theoretical argument, the role
played by other Bt toxin receptors. In fact, a vast literature de-
scribes the involvement of several membrane-bound proteins,
including aminopeptidase N and, more recently, alkaline phospha-
tase, as well as glycolipids (Griftts et al., 2005), in toxin binding
and pore formation (Gmez et al., 2007; Likitvivatanavong et al.,
2011; Pigott and Ellar, 2007).
Finally, according to the signaling pathway model, specic
binding of an active toxin to its cadherin receptor sufces to kill
the cell. The authors again simply ignore studies describing Cry
toxin mutants that have retained an unaltered binding ability,
but completely lost their toxicity (Girard et al., 2009; Kumar and
Aronson, 1999; Tigue et al., 2001). One could argue that although
such mutants bind to the membrane, their specic interaction with
the cadherin is altered in such a way that the signaling pathway
does not become activated. Two of the above-cited references,
however, document that binding of an excess amount of either
one of several inactive mutants is sufciently specic to prevent
binding or pore formation by the corresponding wild-type toxin
(Girard et al., 2009; Tigue et al., 2001).
In summary, the signaling pathway model is not easily tenable,
especially in that it negates the role of pore formation in the mech-
anism of action of Bt toxins that have long been shown to have the
capacity to permeabilize efciently their target membrane in the
insect midgut epithelium, as well as in articial membranes. It
has the merit, however, of stressing the fact that the interaction
of the toxins with sensitive cells undoubtedly has important conse-
quences on cellular metabolism and its regulation. Indeed, earlier
studies, that have been largely ignored, have demonstrated strong
effects of insecticidal Cry toxins on intracellular calcium levels
(Monette et al., 1997, 1994; Potvin et al., 1998; Schwartz et al.,
1991), indicative not only of plasma membrane permeabilization,
but also of intracellular signaling. In fact, parasporin-1, a Bt Cry
toxin specically active against certain cancer cells, was recently
shown to cause intracellular calcium levels to increase in toxin-
sensitive cells (Katayama et al., 2007). In addition, several recent
studies have implicated a variety of intracellular signaling path-
ways in cellular defense mechanisms against nematocidal (Bellier
et al., 2009; Bischof et al., 2008; Chen et al., 2010; Huffman et al.,
2004) and insecticidal (Cancino-Rodezno et al., 2010; Tanaka
et al., 2012) Cry toxins. Clearly, more attention needs to be devoted
to the alterations caused by the toxins, and the pores they form in
the plasma membrane, to the target cells signaling and regulatory
pathways (Kao et al., 2011).
4. Conclusions
During the last several years, the signaling pathway model and,
more importantly, the sequential binding model have attracted
considerable attention and generated an abundant literature.
Unfortunately, however, a careful evaluation of the available data
reveals that both of these models are supported by rather little reli-
able experimental evidence. Many important questions concerning
the mechanism by which insect cells are killed by Bt toxins remain
just as poorly understood as they were before these models were
put forward. For instance, even though a pre-pore structure consti-
tutes a central feature of the sequential binding model, the crucial
question of whether the pores are assembled before or after the
toxin inserts into the membrane is still far from being denitively
resolved. On the other hand, although several intracellular signal-
ing pathways appear to be activated in susceptible cells by Cry tox-
ins, the mechanisms by which these pathways contribute to the
pathogenesis or protect against the deleterious effects of the toxins
remain practically unexplored.
The signaling pathway model is poorly supported by experi-
mental evidence and the components added to the classical activa-
tion ?binding ?pore formation ?lysis model in the sequential
binding model are far from being necessary, as claimed, to describe
the mode of action of Cry toxins. Their usefulness as models needs
to be conrmed by additional independent experimental studies.
Ultimately, the classical, simple model proposed in the 1980s
and the 1990s remains the most valid framework available to ori-
ent future studies aiming at the elucidation of Bt toxins mode of
action.
Acknowledgments
This work was supported in part by grants from the Natural Sci-
ences and Engineering Research Council of Canada, the Fonds de la
recherche en sant du Qubec and the Fonds de recherche du Qu-
bec Nature et technologies. We also thank several colleagues for
constructive suggestions on the manuscript.
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