Insect Control: Biological and Synthetic Agents - Index of

ilayers (Lorence et al., 1995; Peyronnet et al., 2002)
and the estimation of pore size at 10–20 A ˚ (Von-
Tersch et al., 1994), it has been proposed that the
pore could be formed by an oligomer of Cry toxins
containing four to six toxin monomers. Moreover,
intermolecular interaction between Cry1Ab toxin
monomers is a necessary step for pore formation
and toxicity (Soberón et al., 2000). This conclusion
was derived from studies that used two Cry1Ab
mutant proteins that affected different steps in toxicity
(binding and pore formation). Individually
these mutant proteins had decreased toxicity to
M. sexta; however, when assayed as a mixture of
the two toxins, pore formation activity and toxicity
against M. sexta larvae was recovered. These results
show that monomers affected in different steps of
their mode of action can form functional heterooligomers,
and that oligomerization is a necessary step
for toxicity (Soberón et al., 2000). Recently, the
structure of the pore formed by Cry1Aa toxin was
analyzed by atomic force microscopy showing that
the pore is a tetramer (Vie et al., 2001). These data
are in agreement with the proposition of a prepore
structure composed of four monomers (Gómez et al.,
2002b). The regions of the toxin involved in oligomerization
have not been determined; however,
based on mutagenesis studies and analysis of toxin
aggregation it has been suggested that some residues
of helixa-5 may be implicated in this process
(Aronson et al., 1999; Vie et al., 2001).
The pore activity of Cry toxins has been studied
by a variety of electrophysiological techniques
(Schwartz and Laprade, 2000), for example using
synthetic membranes without receptor or in isolated
brush border membrane vesicles containing natural
receptors (Lorence et al., 1995; Peyronnet et al., 2001,
2002; Bravo et al., 2002a). Also ion channels induced
by various activated Cry1 toxins in its monomeric
form – Cry1Aa (Grochulski et al., 1995; Schwartz
et al., 1997a), Cry1Ac (Slatin et al., 1990; Schwartz
et al., 1997a; Smedley et al., 1997), and Cry1C
(Schwartz et al., 1993; Peyronnet et al., 2002) – have
been analyzed in black lipid bilayers. The channel
formation of these toxins was extremely inefficient
and in some studies was only achieved mechanically
(Peyronnet et al., 2001, 2002). The toxin concentrations
needed to achieve channel formation in these
conditions were two to three orders of magnitude
higher than their in vivo insecticidal concentration.
Conductance varied from 11 to 450 pS and multiple
subconducting states are frequently observed
showing unstable traces with current jumps of intermediate
levels that are difficult to resolve. These
high conductances are probably related to clusters of
various numbers of identical size pores operating
7: Bacillus thuringiensis: Mechanisms and Use 263
synchronously rather than pore oligomer structures
of different sizes (Peyronnet et al., 2002). Under nonsymmetrical
ionic conditions, the shift in the reversal
potential (zero current voltage Erev) towardstheK þ
equilibrium potential (E K) indicated that channels
of Cry1 toxins are slightly cation selective. In fact,
several reports indicated that Cry toxins form pores
that are poorly selective to cationic ions including
divalent cations (Lorence et al., 1995; Kirouac et al.,
2002). As mentioned previously, the presence of
receptor (APN) diminished the concentration, more
than 100-fold, of Cry1Aa toxin required for pore
formation activity in synthetic planar bilayers
(Schwartz et al., 1997a). Studies performed in lipid
bilayers containing fused brush border membrane
vesicles isolated from the target insect suggested that
the channels formed by Cry1 toxins in the presence
of their receptors have higher conductance than those
formed in receptor free bilayers. The conductance of
monomeric Cry1C induced channels ranged from
50 pS to 1.9 nS in bilayers containing brush border
membrane vesicles from S. frugiperda (Lorence et al.,
1995). Similarly, the conductance of channels induced
by the monomeric form of Cry1Aa toxin in bilayers
containing membranes from L. dispar were about
eightfold larger than the channels formed in the absence
of receptor (Peyronnet et al., 2001). However,
the presence of multiple conductances is still observed
and the instability of the currents induced in these
studies suggested that even in the presence of receptors
the insertion of monomers into the membrane
does not involve a single conformation. In contrast,
preliminary analysis of the currents induced by pure
oligomer preparations in the absence of receptor
showed highly stable conductance, suggesting a stable
insertion of a single conformation of the toxin into the
membrane (Muñoz-Garay and Bravo, unpublished
data). Finally it is important to mention that, in contrast
to other pore forming toxins, the pore formation
activity of Cry1 proteins is not regulated by low pH,
suggesting that Cry toxins are not internalized into
acidic vesicles for insertion as other pore forming
toxins (Tran et al., 2001).
Overall, the mode of action of Cry toxins can be
visualized as follows (Figure 8):
1. Solubilization of the crystal and activation of the
protoxin by midgut proteases resulting in the
monomer toxin production.
2. Binding of the monomer to the cadherin receptor
located in the apical membrane of midgut cells,
probably accompanied by a mild denaturation
of the monomer that allows proteolytic cleavage
of helix a-1. This cleavage might result in a
conformational change and the formation of a