Insect Control: Biological and Synthetic Agents - Index of

8: Mosquitocidal B. sphaericus: Toxins, Genetics, Mode of Action, Use, and Resistance Mechanisms 293
Mtx1 was initially measured using fusion proteins
synthesized in E. coli: Mtx1 is highly active against
C. quinquefasciatus larvae (Thanabalu et al.,
1992), as the LC50 value is comparable to that of
the crystal Bin proteins (15 ng/ml). Deletion analysis
suggested that the 27 kDa fragment of Mtx1 is able
to self ADP-ribosylate, whereas the 70 kDa fragment
is assumed to play a role in the toxicity to
cultured C. quinquefasciatus cells; however, both
regions are necessary for toxicity to mosquito larvae
(Thanabalu et al., 1993).
Due to its cytotoxicity towards bacterial cells,
the 27 kDa enzyme fragment cannot be produced
alone, in the activated form, in E. coli expression
systems. However, a nontoxic 32 kDa N-terminal
truncated version of Mtx1 has been successfully
expressed in E. coli and subsequently cleaved to an
active 27 kDa enzyme fragment (Schirmer et al.,
2002b). In vitro, this 27 kDa fragment of Mtx1 is
able to ADP-ribosylate numerous proteins in E. coli
lysates, especially a 45 kDa protein identified as the
E. coli elongation factor, Tu (EF-Tu). The inactivation
of EF-Tu by Mtx1-mediated ADP-ribosylation
and the resulting inhibition of bacterial protein
synthesis probably play important roles in the cytotoxicity
of the 27 kDa enzyme fragment of Mtx1
towards E. coli (Schirmer et al., 2002b). Glu 197 of
the 27 kDa enzyme component was identified as
the ‘‘catalytic’’ glutamate that is conserved in all
ADP-ribosyltransferases (Schirmer et al., 2002a).
Transfection of mammalian HeLa cells with a vector
encoding the 27 kDa fragment fused with a green
fluorescent protein leads to cytotoxic effects
characterized by cell rounding and formation of
filopodia-like protrusions (Schirmer et al., 2002a).
In vitro binding assays using isolated BBMFs
from C. pipiens midguts indicate the presence of
a membrane receptor to the 70-kDa fragment. The
affinity of this receptor/toxin interaction is low (Kd
1.4 mM), indicating that the 70-kDa component
has a very low affinity for its binding site (Figure 6a;
Charles and Berry, unpublished data). In addition,
saturation of this binding site was not possible.
Complementary competition experiments, using either
the labeled P70 or the labeled Bin toxin as the
labeled component, and unlabeled P70 or Bin toxin
as the unlabeled competitors, showed that the Bin
toxin can compete with the labeled P70 component,
whereas the opposite is not true (Figure 6b and c).
This suggests that the P70-Mtx1 component shares
a common binding site with the Bin toxin, and that
the Bin toxin can also bind to this binding site with a
very low affinity.
After cleavage of Mtx1 by chymotrypsin-like
proteases, the P70 proteolytic fragment of Mtx1
remains noncovalently bound to the N-terminal
27-kDa fragment (Schirmer et al., 2002a). Thus,
from a functional point of view, the binding of the
P70 component of the toxin to a membrane receptor
may play a key role in carrying the 27-kDa
enzymatic fragment to its target site in mosquito
midgut cells.
Figure 6 Direct binding of 125 I-labeled B. sphaericus P70-Mtx1 toxin to midgut brush border membranes of C. pipiens (a). Inset,
specific binding in Scatchard plots. Competition experiments between 125 I-P70 and unlabeled P70 or Bin toxin (b), or between
125 I-Bin toxin and unlabeled P70 or Bin toxin (c).