Insect Control: Biological and Synthetic Agents - Index of

256 7: Bacillus thuringiensis: Mechanisms and Use
lytic pores in microvilli apical membranes (Schnepf
et al., 1998; Aronson and Shai, 2001). Cell lysis and
disruption of the midgut epithelium releases the cell
contents providing spores a rich medium that is
suitable for spore germination leading to a severe
septicemia and insect death (Schnepf et al., 1998;
de Maagd et al., 2001).
7.4.2. Solubilization and Proteolytic
Activation
Solubilization of long protoxins (130 kDa) depends
on the highly alkaline pH that is present in guts of
lepidopteran and dipteran insects, in contrast to
coleopteran insect guts that have a neutral to slightly
acidic pH (Dow, 1986). In a few cases, protoxin
solubilization has been shown to be a determinant
for insect toxicity. Cry1Ba is toxic to the coleopteran
L. decemlineata only if the protoxin is previously
solubilized in vitro, suggesting insolubility of the
toxin at the neutral–acidic pH of coleopteran insects
(Bradley et al., 1995). The C-terminal portion of
protoxins contains many cysteine residues that
form disulfide bonds in the crystal inclusions and,
therefore, reducing the disulfide bonds is a necessary
step for the solubilization of long Cry protoxins (Du
et al., 1994). Differences in the midgut pH between
lepidopteran and coleopteran midguts may be a
reason for the bias in the utilization of arginine as
basic amino acid over lysine in the lepidopteran
specific toxins Cry1, Cry2, and Cry9 with exception
of the Cry1I toxin, which is also active against coleopteran
insects (Grochulski et al., 1995; de Maagd
et al., 2001). The higher pKa of arginine, compared
with that of lysine, might be required for maintaining
a positive charge even at the high pH of
lepidopteran guts (up to pH 11) resulting in soluble
toxins at alkaline pH.
Proteolytic processing of Cry toxins is a critical
step involved not only on toxin activation but also
on specificity (Haider and Ellar, 1989; Haider et al.,
1989) and insect resistance (Oppert et al., 1997;
Shao et al., 1998). Besides pH, lepidopteran and
coleopteran insects differ in the type of proteases
present in the insect gut; serine proteases are the
main digestive proteases of Lepidoptera and Diptera,
whereas cysteine and aspartic proteases are
abundant in the midguts of Coleoptera (Terra and
Ferreira, 1994). It has been reported that enhanced
degradation of Cry toxins is associated with the loss
of sensitivity of fifth instar Spodoptera litoralis larvae
to Cry1C (Keller et al., 1996) and that serine
protease inhibitors enhanced the insecticidal activity
of some B. thuringiensis toxins up to 20-fold
(MacIntosh et al., 1990). More recently, it was
found that the low toxicity of Cry1Ab toxin to
S. frugiperda could be explained in part by rapid
degradation of the toxin on the insect midgut
(Miranda et al., 2001). For several Cry proteins
inactivation within the insect gut involves intramolecular
processing of the toxin (Choma et al., 1990;
Lambert et al., 1996; Audtho et al., 1999; Pang
et al., 1999; Miranda et al., 2001). However, for
several other Cry toxins, intramolecular processing
is not always related to loss of toxicity and sometimes
is required for proper activation of the toxin
(Dai and Gill, 1993; Zalunin et al., 1998; Yamagiwa
et al., 1999). Therefore, in some cases, differential
proteolytic processing of Cry toxins in different
insects could be a limiting step in the toxicity of
Cry proteins (Miranda et al., 2001).
One interesting feature of Cry toxin activation is
the processing of the N-terminal end of the toxins.
The three-dimensional structure of Cry2Aa protoxin
showed that two a-helices of the N-terminal
region occlude a region of the toxin involved in the
interaction with the receptor (Morse et al., 2001)
(Figure 3). Several lines of evidences suggest that the
processed N-terminal peptide of Cry protoxins
might prevent binding to nontarget membranes
(Martens et al., 1995; Kouskoura et al., 2001;
Bravo et al., 2002b). Escherichia coli cells producing
Cry1Ab or Cry1Ca toxins lacking the
N-terminal peptide were severely affected in growth
(Martens et al., 1995; Kouskoura et al., 2001). It
was speculated that the first 28 amino acids prevented
the Cry1A toxin from inserting into the
membrane (Martens et al., 1995; Kouskoura et al.,
2001). Recently, it was found that a Cry1Ac mutant
that retains the N-terminus end after trypsin treatment
binds nonspecifically to Manduca sexta membranes
and was unable to form pores on M. sexta
brush border membrane vesicles (Bravo et al.,
2002b). Therefore, processing of the N-terminal
end of Cry protoxins may unmask a hydrophobic
patch of the toxin involved in toxin–receptor or
toxin–membrane interaction (Morse et al., 2001;
Bravo et al., 2002b).
7.4.3. Receptor Identification
The major determinant of Cry toxin selectivity is the
interaction with specific receptors on the insect gut
of susceptible insects (Jenkins and Dean, 2000).
Therefore, receptor identification is fundamental
for determining the molecular basis of Cry toxin
action and also in insect resistance management
that in many cases has been shown to correlate
with defects in receptor binding (Ferré and Van
Rie, 2002). A number of putative receptor molecules
for the lepidopteran specific Cry1 toxins have
been identified. In M. sexta, the Cry1Aa, Cry1Ab,