ARTICLE IN PRESS

Toxicon 49 (2007) 423–435

www.elsevier.com/locate/toxicon

Review

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and

their potential for insect control
Alejandra Bravoa, Sarjeet S. Gillb, Mario Soberóna,
a
Departamento de Microbiologı´a Molecular, Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Apdo, Postal 510-3,
Cuernavaca, Morelos 62250, Mexico
b
Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA
Received 18 September 2006; accepted 17 November 2006
Available online 30 November 2006

Abstract

Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity
against insects of different orders—Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as
nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores.
Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode
of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry
family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other
pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface
and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure
that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane.
Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-
membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and
compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect
pests and insect vectors of human diseases.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Cry; Cyt; Bacillus thuringiensis; Receptor; Mode of action; Synergism

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
2. Diversity, structure and evolution of Cry toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
3. Mode of action of three-domain Cry toxins in lepidopteran insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
3.1. Receptor binding in lepidopteran larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
3.2. Pre-pore formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
3.3. Membrane insertion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428

Corresponding author. Tel.: +52 777 3291618; fax: +52 777 3291624.
E-mail address: mario@ibt.unam.mx (M. Soberón).

0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2006.11.022
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424 A. Bravo et al. / Toxicon 49 (2007) 423–435

4. Mode of action of Cry and Cyt toxins in mosquitoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429

4.1. Receptor binding in mosquito midgut membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
4.2. Synergism of Cyt and Cry toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
5. Applications of Cry toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
6. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

1. Introduction receptor binding inducing the formation of an

oligomeric structure that is insertion competent.
Bacillus thuringiensis (Bt) are gram-positive Finally, membrane insertion is triggered, in most
spore-forming bacteria with entomopathogenic cases, by a decrease in pH that induces a molten
properties. Bt produce insecticidal proteins during globule state of the protein (Parker and Feil, 2005).
the sporulation phase as parasporal crystals. These
crystals are predominantly comprised of one or 2. Diversity, structure and evolution of Cry toxins
more proteins Crystal (Cry) and Cytolitic (Cyt)
toxins, also called d-endotoxins. Cry proteins are Cry proteins are specifically toxic to the insect
parasporal inclusion (Cry) proteins from Bt that orders Lepidoptera, Coleoptera, Hymenoptera and
exhibit experimentally verifiable toxic effect to a Diptera, and also to nematodes. In contrast, Cyt
target organism or have significant sequence simi- toxins are mostly found in Bt strains active against
larity to a known Cry protein. Similarly, Cyt Diptera. The Cry proteins comprise at least 50
proteins are parasporal inclusion proteins from Bt subgroups with more than 200 members. Cry
that exhibits hemolytic (Cyt) activity or has obvious proteins are defined as: a parasporal inclusion
sequence similarity to a known Cyt protein. These protein from Bt that exhibits toxic effects to a
toxins are highly specific to their target insect, are target organism, or any protein that has obvious
innocuous to humans, vertebrates and plants, and sequence similarity to a known Cry protein (Crick-
are completely biodegradable. Therefore, Bt is a more et al., 1998). Cyt toxins are included in this
viable alternative for the control of insect pests in definition but it was agreed that proteins that are
agriculture and of important human disease vectors structurally related to Cyt toxins retain the mne-
(Bravo et al., 2005). monic Cyt (Crickmore et al., 1998). Primary
Bt Cry and Cyt toxins belong to a class of sequence identity among different gene sequences
bacterial toxins known as pore-forming toxins is the bases of the nomenclature of Cry and Cyt
(PFT) that are secreted as water-soluble proteins proteins. Additionally, other insecticidal proteins
undergoing conformational changes in order to that are not related phylogenetically to the three-
insert into, or to translocate across, cell membranes domain Cry family have been identified. Among
of their host. There are two main groups of PFT: these, are binary-like toxins and Mtx-like toxins
(i) the a-helical toxins, in which a-helix regions form related to B. sphaericus toxins, and parasporins
the trans-membrane pore, and (ii) the b-barrel produced by Bt (Crickmore et al., 1998).
toxins, that insert into the membrane by forming a The members of the three-domain family, the
b-barrel composed of b-sheet hairpins from each larger group of Cry proteins, are globular molecules
monomer (Parker and Feil, 2005). The first class of containing three structural domains connected by
PFT includes toxins such as the colicins, exotoxin single linkers. One particular feature of the members
A, diphtheria toxin and also the Cry three-domain of this family is the presence of protoxins with two
toxins. On the other hand, aerolysin, a-hemolysin, different lengths. One large group of protoxins is
anthrax protective antigen, cholesterol-dependent approximately twice as long as the majority of the
toxins as the perfringolysin O and the Cyt toxins toxins. The C-terminal extension found in the long
belong to the b-barrel toxins (Parker and Feil, protoxins is dispensable for toxicity and is believed
2005). In general, PFT-producing bacteria secrete to play a role in the formation of the crystal
their toxins and these toxins interact with specific inclusion bodies within the bacterium (de Maagd
receptors located on the host cell surface. In most et al., 2001). Cyt toxins comprise two highly
cases, PFT are activated by host proteases after related gene families (Cyt1 and Cyt2) (Crickmore
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A. Bravo et al. / Toxicon 49 (2007) 423–435 425

et al., 1998). Cyt toxins are also synthesized as case of domain II, structural similarities with several
protoxins and small portions of the N-terminus carbohydrate-binding proteins like vitelline, lectin
and C-terminus are removed to activate the toxin jacalin, and lectin Mpa have been reported (de
(Li et al., 1996). Maagd et al., 2003). Domain III, shares structural
To date, the tertiary structures of six different similarity with other carbohydrate-binding proteins
three-domain Cry proteins, Cry1Aa, Cry2Aa, such as the cellulose binding domain of 1,4-b-
Cry3Aa, Cry3Bb, Cry4Aa and Cry4Ba have been glucanase C, galactose oxidase, sialidase, b-glucor-
determined by X-ray crystallography (Fig. 1) (Li onidase, the carbohydrate-binding domain of xyla-
et al., 1991; Grochulski et al., 1995; Morse et al., nase U and b-galactosidase (de Maagd et al., 2003).
2001; Galitsky et al., 2001; Boonserm et al., 2005, These similarities suggest that carbohydrate moi-
2006). All these structures display a high degree of eties could have an important role in the mode of
similarity with a three-domain organization, sug- action of three-domain Cry toxins. Interestingly, in
gesting a similar mode of action of the Cry three- the nematode C. elegans, mutations in bre genes
domain protein family. The N-terminal domain involved in the synthesis of certain glycolipids lead
(domain I) is a bundle of seven a-helices in which to Cry5 resistance showing that glycolipids are
the central helix-a5 is hydrophobic and is encircled important receptor molecules of Cry5 (Griffits et al.,
by six other amphipathic helices; and this helical 2005).
domain is responsible for membrane insertion and Cyt proteins, on the other hand, have a single a–b
pore-formation. Domain II consists of three anti- domain comprising of two outer layers of a-helix
parallel b-sheets with exposed loop regions, and hairpins wrapped around a b-sheet (Li et al., 1996,
domain III is a b-sandwich (Li et al., 1991; Fig. 1). Cyt toxin is structurally related to volvatox-
Grochulski et al., 1995; Morse et al., 2001; Galitsky in A2, a PFT cardiotoxin produced by a straw
et al., 2001; Boonserm et al., 2005, 2006). Exposed mushroom Volvariella volvacea (Lin et al., 2004).
regions in domains II and III are involved in An analysis of the phylogenetic relationships of
receptor binding (Bravo et al., 2005). Domain I the isolated domains of members of the three-
shares structural similarities with other PFT like domain Cry family revealed interesting features
colicin Ia and N and diphtheria toxin, supporting regarding the creation of diversity in this protein
the role of this domain in pore-formation. In the family (Bravo, 1997; de Maagd et al., 2001).

Fig. 1. Three-dimensional structures of insecticidal toxins produced by Bacillus thuringiensis Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa,
Cry4Bb and Cyt2A.
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426 A. Bravo et al. / Toxicon 49 (2007) 423–435

Domains I and II have coevolved. The analysis of Protoxin size

domain III sequences, revealed a different topology Cry1A 130 kDa
due to the fact that several examples of domain III 29 51 605

swapping among toxins occurred (Bravo, 1997; de

Maagd et al., 2001). Some toxins with dual Cry3A 67 kDa
58 643
specificity (coleopteran, lepidopteran) are clear
examples of domain III swapping among coleopter- Cry4B 130 kDa
an and lepidopteran specific toxins. This suggests 43 203 679

that domain III swapping could create novel

specificities. In this regard, in vitro domain III Cry11A 70 kDa
28 361 643
swapping of certain Cry1 toxins resulted in changes
in insect specificity (Bosch et al., 1994; de Maagd Fig. 2. Relative length of Cry protoxins and position of protease
et al., 2000). The independent evolution of the three digestion. White boxes represent the protoxin and striped boxes
represent the activated toxin. Solid arrows show the amino- and
structural domains and domain III swapping among
carboxy- terminal cleavage sites of the activated toxins. Doted
different toxins generated proteins with similar arrows show the intramolecular cleavages. Cleavage of Cry1A at
mode of action but with very different specificities residue 51 resulted in loss of helix a-1 and pre-pore formation.
(Bravo, 1997; de Maagd et al., 2001). Cleavage of Cry4B resulted in two fragments of 18 and 46 kDa,
while Cry11A resulted in two fragments of 34 and 32 kDa.
3. Mode of action of three-domain Cry toxins in
lepidopteran insects (Aronson and Shai, 2001; Bravo et al., 2005).
Subsequently cell lysis and disruption of the midgut
The mode of action of Cry toxins has been epithelium releases the cell contents providing
characterized principally in lepidopteran insects. As spores a germinating medium leading to a severe
mentioned previously, it is widely accepted that the septicemia and insect death (de Maagd et al., 2001;
primary action of Cry toxins is to lyse midgut Bravo et al., 2005).
epithelial cells in the target insect by forming One interesting feature of Cry toxin activation is
pores in the apical microvilli membrane of the cells the processing of the N-terminal end of the toxins.
(Aronson and Shai, 2001; de Maagd et al., 2001, The three-dimensional structure of Cry2Aa proto-
Bravo et al., 2005). Nevertheless, it has been xin showed that two a-helices of the N-terminal
recently suggested that toxicity could be related to region occlude a region of the toxin involved in the
G-protein mediated apoptosis following receptor interaction with the receptor (Morse et al., 2001).
binding (Zhang et al., 2006). Cry proteins pass from Also, it was found that a Cry1Ac mutant that
crystal inclusion protoxins into membrane-inserted retained the N-terminus end after trypsin treatment
oligomers that cause ion leakage and cell lysis. The binds nonspecifically to Manduca sexta membranes
crystal inclusions ingested by susceptible larvae and was unable to form pores on M. sexta brush
dissolve in the alkaline environment of the gut, border membrane vesicles (BBMV) (Bravo et al.,
and the solubilized inactive protoxins are cleaved by 2002). Therefore, processing of the N-terminal end
midgut proteases yielding 60–70 kDa protease re- of Cry protoxins may unmask a domain II
sistant proteins (Bravo et al., 2005). Toxin activa- hydrophobic patch involved in toxin–receptor or
tion involves the proteolytic removal of an N- toxin–membrane interaction.
terminal peptide (25–30 amino acids for Cry1
toxins, 58 residues for Cry3A and 49 for Cry2Aa) 3.1. Receptor binding in lepidopteran larvae
and approximately half of the remaining protein
from the C-terminus in the case of the long Cry For Cry1A toxins, at least four different binding-
protoxins. Fig. 2 shows a schematic representation proteins have been described in different lepidop-
of the Cry protoxin structure and their protease teran insects; a cadherin-like protein (CADR),
cleavege sites. The activated toxin then binds to a glycosylphosphatidyl-inositol (GPI)-anchored
specific receptors on the brush border membrane of aminopeptidase-N (APN), a GPI-anchored alkaline
the midgut epithelium columnar cells (de Maagd phosphatase (ALP) and a 270 kDa glycoconjugate
et al., 2001; Bravo et al., 2005) before inserting into (Vadlamudi et al., 1995; Knight et al., 1994; Jurat-
the membrane. Toxin insertion leads to the forma- Fuentes and Adang, 2004; Valaitis et al., 2001).
tion of lytic pores in microvilli of apical membranes Fig. 3 shows a representation of the four types of
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A. Bravo et al. / Toxicon 49 (2007) 423–435 427

reported (Hua et al., 2004). In the case of the

Heliothis virescens CADR, site-directed mutagenesis
GalNAc GalNAc narrowed this binding region to residues 1422–1440
and shown to bind Cry1Ac domain II loop 3
(b10–b11 loop) (Xie et al., 2005).
The most frequent mechanism of resistance to
Cry toxins involves a change in receptor binding
(Ferré and van Rie, 2002). In the case of the
APN ALP laboratory selected H. virescens Cry1Ac-resistant
CADR GCR line YHD2, it was shown that a single mutation was
responsible for 40–80% of Cry1Ac resistance levels
Fig. 3. Receptor molecules of Cry1A proteins. CADR, cadherin
receptor; APN, aminopeptidase-N, ALP, alkaline phosphatase,
and shown to be linked to a retrotransposon
GCR, 270 kDa glyco-conjugate receptor. insertion in the CADR gene (Gahan et al., 2001).
Also, characterization of CADR alleles in field-
derived and laboratory selected strains of Pectino-
putative Cry1A-receptor molecules characterized so phora gossypiella (pink bollworm) and Helicoverpa
far. The role of toxin–receptor interaction has been armigera revealed different mutated CADR alleles
particularly well described in M. sexta. In this that were associated with Cry1Ac resistance (Morin
insect, at least two Cry1A-binding proteins, a et al., 2003; Xu et al., 2005).
CADR protein (Bt-R1) and a GPI-anchored APN, Regarding APN, Cry1Ac toxin binds to APN
have been described as receptors (Vadlamudi et al., receptor by means of domain III that specifically
1995; Knight et al., 1994). Cadherins are transmem- recognizes N-acetylgalactosamine (GalNAc) moi-
brane proteins with a cytoplasmic domain and an eties in contrast to Cry1Aa and Cry1Ab toxins that
extracellular ectodomain with several cadherin show no GalNAc-binding capacities (Masson et al.,
repeats, 12 in the case of Bt-R1 (Vadlamudi et al., 1995). Based on the use of monoclonal antibodies
1995). The ectodomain contains calcium-binding that competed binding of Cry1Aa with Bombyx
sites, integrin interaction sequences and cadherin- mori APN, the Cry1Aa–APN interacting epitopes
binding sequences. Surface plasmon resonance were recently mapped in domain III b16
experiments showed that the binding affinity of (508STLRVN513) and b22 (582VFTLSAHV589) resi-
monomeric Cry1A toxins to the M. sexta Bt-R1 is in dues (Atsumi et al., 2005). Surface plasmon
the range of 1 nM (Vadlamudi et al., 1995), while resonance binding studies of Cry1Ab mutants with
that of APN is in the range of 100 nM (Jenkins and pure M. sexta APN showed that domain II loop 2
Dean, 2000). and loop 3 are also involved in APN recognition
The interaction of Cry1A toxins with the Bt-R1 (Jenkins and Dean, 2000). In the case of the
receptor is rather complex involving at least three lepidopteran insect L. dispar, a sequential-binding
binding epitopes in the two molecules. Using a mechanism was proposed in the interaction of
synthetic phage-antibody library, Gómez et al. (2001, Cry1Ac with APN (Jenkins et al., 2000). Cry1Ac
2002a) characterized an scFv antibody (scFv73) that domain III first interacts with APN GalNAc sugar
binds to domain II loop 2 (b6–b7 loop) of Cry1A moieties facilitating the subsequent interaction of
toxins. This antibody inhibited binding of Cry1A domain II loop regions with another region in this
toxins to Bt-R1 but not to APN. Sequence analysis of receptor (Jenkins et al., 2000). In addition, it was
the CDR3H region of scFv73 led to the identification described that a Cry1C-resistant Spodoptera exigua
of an eight amino acid epitope in Bt-R1 CADR colony did not express the APN1 (Herrero et al.,
repeat 7 (869HITDTNNK876) involved in toxin 2005).
binding to domain II loop 2 of Cry1A toxins (Gómez In the case of ALP receptor, it was demonstrated
et al., 2001, 2002a). Additionally, another binding that in the H. virescens resistant line YHD2 part of
epitope in Bt-R1 CADR repeat 11 (1331IPLPA- the Cry1Ac-resistant phenotype is due to mutations
SILTVTV1342) that interacts with domain II loop that lower the production of the GPI-anchored ALP
a8 (a8a–a8b loop) and loop 2 of Cry1Ab toxin was receptor (Jurat-Fuentes and Adang, 2004). In
identified (Gómez et al., 2003). Finally, a third region M. sexta, proteomic analysis of BBMV Cry1Ac-
in CADR repeat 12 of Bt-R1 (residues 1363–1464) binding proteins also revealed ALP as a putative
involved in Cry1Ab interaction and toxicity was receptor molecule (McNall and Adang, 2003).
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3.2. Pre-pore formation 3.3. Membrane insertion

As mentioned previously several PFT form a After exposure of BBMV to Cry1A toxins, these
soluble oligomeric structure before membrane toxins were found associated with lipid rafts
insertion. In the case of Cry1Ab toxin, binding of microdomains and it was reported that the integrity
this toxin to M. sexta Bt-R1 promotes an additional of these microdomains was essential for in vitro
proteolytic cleavage in the N-terminal end of Cry1Ab pore-forming activity (Zhuang et al., 2002).
the toxin (helix a-1) facilitating the formation The Bt-R1 receptor is located in soluble membrane
of a pre-pore oligomeric structure that is important in contrast with APN and ALP receptors, which are
for insertion into the membrane and for toxicity attached to the membrane by GPI anchors, and are
(Gómez et al., 2002b; Rausell et al., 2004a). preferentially partitioned into lipid rafts (Zhuang
Incubation of Cry1Ab protoxin with the single et al., 2002). Lipid rafts are detergent-insoluble lipid
chain antibody scFv73 that mimics the CADR microdomains enriched in cholesterol and sphingo-
receptor or with the toxin-binding peptides of lipids, and GPI-anchored proteins (Simons and
Bt-R1 (CADR repeats 7 and 11), and treatment Toomre, 2000). Like their mammalian counterparts,
with M. sexta midgut juice, resulted in toxin H. virescens and M. sexta lipid rafts are enriched in
preparations with the formation of a 250 kDa cholesterol, sphingolipids, and GPI-anchored pro-
oligomer that lacked the helix a-1 of domain I teins (Zhuang et al., 2002). Lipid rafts have been
(Gómez et al., 2002b, 2003). It was reported that implicated in membrane and protein sorting, and in
oligomeric structures of Cry1Ab and Cry1Ac signal transduction (Simons and Toomre, 2000).
increase 100–200-fold their binding affinity to the Also, they have been described as portals for
APN receptor, showing apparent dissociation different viruses, bacteria and toxins. Different
constants of 0.75–1 nM (Gómez et al., 2003; bacterial PFT interact with receptors located in
Pardo-López et al., 2006). The oligomer, in contrast lipid rafts and this is a crucial step in the
to the 60 kDa monomer, was membrane insertion oligomerization and insertion of PFT into the
competent as judged by measuring toxin membrane membrane (Cabiaux et al., 1997). In the case of
insertion using the intrinsic fluorescence of Cry1A toxins, the APN receptor has been impli-
tryptophan residues and also by the analysis of cated in Cry1A toxin insertion, since cleavage of
membrane permeability using black lipid bilayers APN by phosphatidylinositol phospholipase C
(Rausell et al., 2004a). The pore activity of the treatment, that cleaves out the GPI-anchored
Cry1Ab oligomeric structure analyzed in synthetic proteins, substantially decreased the levels of
planar lipid bilayers revealed different kinetic Cry1Ab oligomer in insoluble membranes and
characteristics from the monomeric Cry1Ab reduced drastically the pore-formation activity of
toxin. First, pore formation by pure oligomer the toxin (Bravo et al., 2004). In addition, APN
preparations was observed at much lower toxin incorporation into lipid bilayers enhanced Cry1Aa
concentrations than the monomeric toxin and pore-formation activity (Schwartz et al., 1997).
secondly, the kinetics were different since oligomeric Using tryptophan fluorescence analysis, structural
Cry1Ab showed stable channels that had a high changes observed after binding of the oligomeric
open probability in contrast to the monomeric Cry1Ac toxin to the APN receptor were studied by
toxin that showed an unstable opening pattern analyzing the binding of GalNAc to the Cry1Ac
(Rausell et al., 2004a). The formation of Cry toxin, since GalNac is a binding determinant in the
oligomeric structures has been demonstrated for Cry1Ac–APN interaction (Pardo-López et al.,
Cry1Aa, Cry1Ab, Cry1Ca, Cry1Da, Cry1Ea, 2006). The in vitro interaction of GalNAc with
Cry1Fa and Cry3 toxins (Gómez et al., 2002b; oligomeric Cry1Ac induced a conformational
Rausell et al., 2004a, 2004c; Muñoz-Garay et al., change in the toxin and enhanced its insertion into
2006). In all cases, the Cry toxin samples containing lipid membranes indicating that the interaction
oligomeric structures correlated with high pore of the pre-pore oligomer of Cry1A toxins with
activity, in contrast to monomeric samples that APN is important for facilitating membrane inser-
showed marginal pore-formation activity, support- tion (Pardo-López et al., 2006).
ing the hypothesis that oligomer formation is a It has been proposed that proteins must partially
necessary step in the mechanism of action of Cry unfold to facilitate membrane insertion and channel
toxins. formation. In the case of PFT active against
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A. Bravo et al. / Toxicon 49 (2007) 423–435 429

mammalian cells, unfolding is triggered by acidic and Cry4B share similar structures with the
pH (Parker and Feil, 2005). Acidic pH could be lepidopteran active toxin Cry1Aa suggesting a
encountered upon cell internalization of the toxins similar mode of action of these Cry proteins in
in acidic membrane compartments, and also in the mosquitoes.
membrane surface that has an acidic pH that could As in lepidopteran insects, in mosquitoes the
be up to 2 pH units lower than the bulk pH (Parker crystals ingested by susceptible larvae dissolve in the
and Feil, 2005). Interestingly, lepidopteran and alkaline gut environment, releasing soluble proteins.
dipteran insects have a basic pH (up to pH 11) in In the case of the 70 kDa Cry11Aa protoxin,
their midgut lumen (Dow, 1986). Unfolding ana- proteolytic activation using gut extract removes 28
lyses of pure Cry1Ab structures at different pHs residues from the N-terminal and cleaves intramo-
demonstrated that the molten globe state of the pre- lecularly resulting in 34 and 32 kDa fragments (Dai
pore complex was induced by alkaline pH (Rausell and Gill, 1993), but these two fragments remain
et al., 2004b). These analyses also showed that the associated and retain toxicity (Fig. 2) (Yamagiwa
pre-pore and membrane inserted oligomer, have a et al., 2004). This is also the case for the 130 kDa
more flexible conformation than the monomeric Cry4Ba protoxin where N-terminal, C-terminal and
toxin (Rausell et al., 2004b). Although not proven, intra-molecular cleavage results in an active toxin
it may be possible that the conformational change comprised of two protein fragments of 18 and
observed after interaction of the pre-pore oligomer 46 kDa (Fig. 2) (Angsuthanasombat et al., 1993;
with APN could be related to molten globule state Komano et al., 1998). As mentioned previously, Cyt
since, as mentioned previously, this interaction toxins are also synthesized as protoxins and small
facilitates membrane insertion (Pardo-López et al., portions of the N-terminus and C-terminus are
2006). Additionally, in the membrane-inserted pore, removed to activate the toxin (Armstrong et al.,
only domain I was protected from heat denatura- 1985, Gill et al., 1987; Li et al., 1996). In the case of
tion, suggesting that it may be inserted into the Cyt2Aa, 32 amino acid residues and 15 amino acid
membrane in contrast to domains II and III residues from the N-terminal and the C-terminal
(Rausell et al., 2004b). Finally, the alkaline pH ends are removed by proteinase K treatment
induced a looser conformation of the membrane- producing a monomeric protein with hemolytic
inserted domain I that is important for an active activity (Koni and Ellar, 1994).
channel formation (Rausell et al., 2004b).
Based on the data described above, we described 4.1. Receptor binding in mosquito midgut membranes
a model involving the sequential interaction of
Cry1A toxins with Bt-R1 and APN receptor It is proposed that Cry toxins bind to specific
molecules. First, the interaction of monomeric protein receptors in the microvilli of the mosquito
Cry1A toxins with Bt-R1 facilitates the formation midgut cells. In contrast, Cyt toxins do not bind to
of a pre-pore oligomeric structure that gains protein receptors but directly interact with mem-
binding-affinity to APN, the pre-pore toxin binds brane lipids inserting into the membrane and
APN, a conformational change occurs and a molten forming pores (Thomas and Ellar, 1983; Gill
globule state of the toxin is induced, the pre-pore is et al., 1987; Li et al., 1996; Promdonkoy and Ellar,
inserted into lipid rafts inducing pore formation and 2003) or destroying the membrane by a detergent
cell swelling (Bravo et al., 2004; Fig. 4A). like interaction (Butko, 2003).
In the mosquitocidal Cry4A, Cry4B and Cry11Aa
4. Mode of action of Cry and Cyt toxins in toxins, domain II loop regions have been implicated
mosquitoes in toxin–receptor interaction and specificity. Cry4-
Ba shows no toxicity to Culex species in contrast to
Bt subsp. israelensis (Bti) is highly toxic to Cry4Aa toxin that is toxic to Culex larvae.
different Aedes, Culex and Anopheles mosquito Mutagenesis of loop 3 region of Cry4Ba to mimic
species that are vectors of human diseases (Marga- that of Cry4Aa introduced toxicity to Culex species
lith and Ben-Dov, 2000). This bacterium produces (Abdullah et al., 2003). In addition, mutagenesis of
crystal inclusions composed of Cry4Aa, Cry4Ba, loops 1 and 2 of Cry4Ba abolished toxicity to Aedes
Cry10Aa, Cry11Aa, Cyt1Aa and Cyt2Ba toxins and Anopheles larvae (Abdullah et al., 2003).
(Berry et al., 2002). As mentioned previously, the Recently, it was demonstrated that redesigning the
mosquitocidal active Cry proteins Cry11Aa, Cry4A Cry1Aa domain II loop amino acid regions to
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430 A. Bravo et al. / Toxicon 49 (2007) 423–435

Fig. 4. Model of the mode of action of Cry and Cyt toxins. (A), sequential interaction of Cry toxins with different receptor molecules in
lepidopteran larvae. (1) Solubilization and activation of the toxin; (2) binding of monomeric Cry toxin to the first receptor (CADR or
GCR), conformational change is induced in the toxin and a-helix 1 is cleaved; (3) oligomer formation; (4) binding of oligomeric toxin to
second receptor (GPI–APN or GPI–ALP), a conformational change occurs and a molten globule state of the toxin is induced; (5) insertion
of the oligomeric toxin into lipid rafts and pore formation. (B), Role of Cyt and Cry toxins in the intoxication of dipteran larvae. (1) Cry
and Cyt toxins are solubilized and activated; (2) Cyt toxin inserts into the membrane and Cry toxin binds to receptors located in the
membrane (ALP or Cyt toxin); (3) oligomerization of the Cry toxin is induced; (4) oligomer is inserted into the membrane resulting in pore
formation.

resemble that of Cry4Ba resulted in a Cry1Aa et al., 2006). However, GPI-anchored proteins
mutant with moderate insecticidal activity against purified after Cry11Aa affinity chromatography
C. pipiens larvae (Liu and Dean, 2006). Also, in the revealed only the 65 kDa. This protein was identi-
case of Cry11Aa toxin, qualitative binding competi- fied as a GPI-anchored ALP enzyme (Fernández
tion assays with synthetic peptides corresponding to et al., 2006). In addition, Ae. aegypti GPI–ALP
predicted Cry11Aa domain II exposed regions protein was shown to be involved in the toxicity of
revealed that loop a-8, b-4 region and loop 3 were Cry11Aa. Two peptide displaying phages,
important for binding to Ae. aegypti BBMV. P1.BBMV and P8.BBMV, that specifically bound
Mutagenesis of putative loop a-8 residues confirmed the 65 kDa ALP, competed with the binding of the
that this region is important for Cry11Aa interaction toxin to BBMV, and interfered with the toxicity of
with Ae. aegypti BBMV and toxicity (Fernández Cry11Aa toxin (Fernández et al., 2006). Overall
et al., 2005). Overall these results show that domain II these results show that the GPI–ALP is an
loop regions are very important for Cry toxin–recep- important receptor molecule that mediates Cry11Aa
tor interaction in mosquitoes. toxicity to Ae. aegypti larvae.
In the case of Cry11Aa and Cry4Ba mosquitoci- In Anopheles quadrimaculatus, a GPI-anchored
dal toxins, binding-proteins of 65 and 62 kDa were APN was identified as a putative Cry11Ba receptor
identified in BBMV from Ae. aegypti larvae by toxin (Abdullah et al., 2006). This APN protein bound
overlay assays (Buzdin et al., 2002). Recent work, Cry11Ba with high affinity but did not interact with
identified three Ae. aegypti midgut proteins of 200, Cry4Ba or Cry11Aa toxins (Abdullah et al., 2006).
100 and 65 kDa that bound Cry11Aa toxin suggest- As in lepidopteran insects, several Cry receptors
ing the presence of multiple receptor molecules for in mosquitoes are attached to the membrane by GPI
Cry toxins in the mosquito membranes. Of these anchors, including ALP and APN (Fernández
proteins, the 100 and 65 kDa were shown to be et al., 2006; Abdullah et al., 2006). Also, in the
anchored to the membrane by GPI (Fernández case of the Bin toxin produced by B. sphaericus, a
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A. Bravo et al. / Toxicon 49 (2007) 423–435 431

GPI-anchored a-glucosidase is the functional re- was determined. The epitopes of Cyt1Aa that bind
ceptor in Culex pipiens (Darboux et al., 2001). As Cry11A were identified. The role of loop b6–aE and
mentioned previously, these and other GPI- part of b7 of Cyt1Aa in binding Cry11Aa was
anchored proteins are proposed to be selectively confirmed by heterologous competition assays using
included in lipid rafts. Therefore it seems that synthetic peptides corresponding to these regions
targeting GPI-anchored proteins may be a general and by site-directed mutagenesis (Pérez et al., 2005).
strategy of PFT, including Cry toxins, to interact Previously, it was suggested that Cyt proteins insert
with their target cells. into the membrane by means of the b-sheets
Further work is needed to determine the role of structures leaving the b6–aE loop exposed (Li
Cry-receptor molecules in mosquitoes and if a pre- et al., 1996). Similarly, the loop a8, b4 and loop 2
pore oligomeric structure is also a membrane- regions of Cry11Aa were identified as the specific
insertion intermediate in these toxins. However, epitopes involved in the binding interaction with
the conserved structure of Cry toxins, the role of Cyt1Aa (Pérez et al., 2005). As mentioned pre-
domain II in receptor interaction and the presence viously, the domain II loop a-8 is involved in
of multiple receptor molecules, some anchored to the interaction of Cry11Aa with ALP receptor
the membrane by GPI, suggests that the mode of (Fernández et al., 2006). Mutagenesis of specific
action of Cry toxins in mosquitoes could be similar amino acid residues in both toxins demonstrated
to that characterized in lepidopteran insects. that binding between these two toxins correlated
Moreover, the role in intracellular signaling, as with synergism (Pérez et al., 2005). These data
observed with the Cry1Ab toxin (Zhang et al., indicate that Cry11Aa binds to membrane-inserted
2006), has to be evaluated for mosquitocidal active Cyt1Aa resulting in the insertion of Cry11Aa into
Cry toxins. the mosquito membranes. This interaction of
Cry11Aa is similar to its binding with its natural
4.2. Synergism of Cyt and Cry toxins receptor. Consequently, this is the first example of
an insect pathogenic bacterium that not only
A major threat to the use of Bt is the appearance produces a toxin but also its functional receptor,
of insect resistance, which has been documented in thereby promoting toxin binding to target mem-
the field with lepidopteran insects (Ferré and van branes and toxicity to the mosquito (Pérez et al.,
Rie, 2002). However, no resistance has been 2005). Fig. 4B shows the proposed mode of action
observed in the field in mosquito species controlled of Cry and Cyt in Ae. aegypti.
with Bti (Becker, 2000). The lack of resistance to Bti
is due to the presence of the Cyt1Aa protein in the 5. Applications of Cry toxins
crystal (Georghiou and Wirth, 1997). Culex quin-
quefasciatus populations resistant to Cry4Aa, Three major applications of Bt toxins have been
Cry4Ba or/and Cry11Aa have been selected under achieved: (i) in the control of defoliator pests in
laboratory conditions but resistance to the Cry forestry, (ii) in the control of mosquitoes that are
proteins could not be selected in the presence of vectors of human diseases, and (iii) in the develop-
Cyt1Aa protein (Wirth et al., 1997). Also, Cyt1Aa ment of transgenic insect-resistant plants.
suppresses the resistance of the Cx. quinquefasciatus One of the most successful applications of Bt has
Cry-resistant populations (Wirth et al., 1997). In been the control of lepidopteran defoliators, which
addition, synergism between Cyt1Aa and the Cry are pests of coniferous forests mainly in Canada and
proteins of Bti has been observed (Chilcott and United States. In both countries, the control of
Ellar, 1988; Angsuthanasombat et al., 1992; Geor- forests defoliators relies mostly on the use of Bt
ghiou and Wirth, 1997); the activity of the Bti strain, HD-1, producing Cry1Aa, Cy1Ab, Cry1Ac
crystals is much higher than that of the isolated and Cry2Aa toxins (van Frankenhuyzen, 2000;
proteins. Recently, it was demonstrated that Bauce et al., 2004). Successful application of Bt is
Cyt1Aa protein synergizes Cry11Aa toxicity by highly dependent on proper timing, weather condi-
functioning as a receptor molecule (Pérez et al., tions and high dosage of spray applications. These
2005). Binding of Cry11Aa to Ae. aegypti BBMV factors combine to determine the probability of
was enhanced by membrane-bound Cyt1Aa and the larvae ingesting a lethal dose (van Frankenhuyzen,
high affinity interaction between Cyt1Aa and 2000; Bauce et al., 2004). The use of Bt in the
Cry11Aa in solution and in membrane bound state control of defoliators has resulted in a significant
 ARTICLE IN PRESS
432 A. Bravo et al. / Toxicon 49 (2007) 423–435

reduction in the use of chemical insecticides for pest insect-resistant crops (Qaim and Zilberman, 2003;
control in the forests. Toenniessen et al., 2003).
As mentioned previously, Bti is highly active
against disease vector mosquitoes like Ae. aegypti 6. Final remarks
(vector of dengue fever), Simulium damnosum
(vector of onchocerciasis) and certain Anopheles The mode of action of Cry toxins is a multi-step
species (vectors of malaria). Its high insecticidal process that involves the interaction with several
activity, the lack of resistance to Bti, the lack receptor molecules leading to membrane insertion
of toxicity to non-target organisms and the appear- and cells lysis. The characterization of the mode of
ance of insect-resistant populations to chemical action of Cry toxins in other susceptible organisms
insecticides resulted in a rapid implementation will be important to fully understand the mode of
of Bti as an alternative control method of action of this family of proteins. Also, the identi-
mosquito and black fly populations (Becker, fication of receptor molecules and binding epitopes
2000). In 1983, a control program for the eradica- will help in the development of strategies to cope
tion of onchocerciasis was launched in eleven with the potential problem of insect resistance. In
countries of Western Africa using Bti since addition, screening of novel Cry proteins with novel
S. damnosum populations had developed resistance insect and receptor specificities will be fundamental
to larvicidal organophosphates (Guillet et al., 1990). for the development of novel products for the
Presently, more than 80% of this region is protected control of different insect pests.
by Bti applications and 20% with the chemical
larvicide, temephos. Furthermore, control of onch-
Acknowledgments
ocerciasis has protected over 15 million children
without the appearance of black fly resistance to Bti
The research work of our groups was supported
(Guillet et al., 1990). This success of vector control
in part by DGAPA/UNAM IN207503-3, IN206
using Bti will certainly increase its use around the
503-3 and IX217404, CONACyT 48631 and 46176-
world. However, the low activity of Bti to certain
Q, USDA 2002-35302-12539 and NIH 1R01 AI0
vector mosquitoes, mainly Anophelines, will require
66014-01.
the isolation of other Bt strains with novel cry genes
more effective against these important disease
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