Quantification of the effect of Bacillus thuringiensis toxins on short-circuit current in the midgut of Bombyx mori

doi:10.1016/0022-1910(94)00093-V

Journal of Insect Physiology

Volume 41, Issue 1, January 1995, Pages 17-22

https://doi.org/10.1016/0022-1910(94)00093-V Get rights and content

Abstract

We examined the abilities of two Bacillus thuringiensis insecticidal toxins, CryIAa and CryIAc, prepared from Escherichia coli-cloned gene products, to inhibit short-circuit current in midgut epithelia of Bombyx mori. Voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short-circuit current (I_SC) by trypsin-activated toxins. Three bathing solutions were compared and the medium of Chamberlin [(1990) J. exp. Biol. 150, 467–471] was found to maintain the highest I_SC for the longest time. For CryIAa, a concentration range between 0.33 and 8.0 ng/ml resulted in inhibition of I_SC at the rates of −0.91 μA/min (lag time, 9 min) and −7.13 μA/min (lag time, 4 min) respectively, showing a correlation between toxin concentration and inhibitory response. Concentrations greater than 1.6 ng/ml showed diminishing additional effects on the I_SC response, indicating an approach to saturation. The lag times decreased with increasing concentration of toxin applied. For CryIAc, the lowest concentration that gave a response was 3.2 ng/ml (slope, −0.31 μA/min; lag time, 2 min). There also was a linear correlation between toxin concentration and response for CryIAc, but effective concentrations of CryIAc were approximately 2 orders of magnitude greater than those of CryIAa.

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Beyond Bacillus thuringiensis: New insecticidal proteins with potential applications in agriculture
2023, Advances in Insect Physiology
Future biotech crops to control agriculturally relevant insects require new modes of action (MoAs) to counter acquired insect resistance or new pest emergence. A new MoA must entail a new “site of action” (SoA) as resistance to insecticidal proteins can involve alteration in the binding site or receptor, a key MoA determinant of insect resistance traits and foundational to discovery of useful new proteins. Bacillus thuringiensis (Bt) has provided nearly all successfully deployed crop incorporated insect protectants, but it has become increasingly difficult to identify new proteins with utility for agricultural applications. Researchers have increasingly turned to ‘non-Bt’ sources that span the biosphere. In this chapter, we present some key insecticidal protein discoveries from bacteria, fungi and plants with differing degrees of promise. These proteins come from a variety of protein structural families, some similar to those identified from Bt, while others are only remotely related and novel. From bacteria, we feature PIP-47Aa (novel), IPD072Aa (novel), AfIP-1A/AfIP-1B (aegerolysin/resembles membrane attack complex perforin-like; MACPF), Mpf2Ba1 (MACPF) and Mpf3Aa1 (MACPF), and from fungi we feature several aegerolysin/MACPF binary proteins. From plants, we cover candidates from lower plants, particularly ferns and lycophytes, drawing a contrast to largely unsuccessful efforts sourcing efficacious genes from higher plants. We feature: Tma12 (AA10 lytic polysaccharide monooxygenases), IPD083 (novel) and the PtIP-50/PtIP-65 binary pair (novel/aerolysin), IPD079 (MACPF), and IPD113 (δ-endotoxin-like). These lower plant discoveries exhibit interesting parallels with microbial insecticidal proteins. From these examples, it becomes clear that non-Bt organisms are rich in proteins with promise for biotech development.
Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review
2012, Journal of Invertebrate Pathology
Citation Excerpt :
This conclusion is very surprising in that it contradicts decades of work in this field. To cite only a few examples, in vitro trypsin-activated monomeric toxins do efficiently 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 insect 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; Kirouac et al., 2006a; Tigue et al., 2001) and inhibit amino acid transport into these vesicles (Sacchi et al., 1986; Wolfersberger, 1991). Moreover, most experiments comparing the permeabilization ability of pre-formed oligomers with that of monomers (Gómez et al., 2002; Muñoz-Garay et al., 2006; Pardo-López et al., 2006; Pérez 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 described by the sequential model.
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 necessary for improving these products and ensuring their continued use. Accordingly, a long history of intensive 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 developed to describe the events leading to membrane insertion and pore formation. However, it was also proposed 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 field 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.
All domains of Cry1A toxins insert into insect brush border membranes
2008, Journal of Biological Chemistry
Citation Excerpt :
The LC50 for each toxin was calculated by probit analysis using SoftTox (WindowsChem Software, Inc.). Voltage Clamp Measurements of Cry1A Mutants—Inhibition of short circuit currents (Isc) was measured by clamping M. sexta midguts using procedures described earlier (28). Briefly, 100 ng of purified proteins were added to the lumen side of the gut stabilized in the buffer (30).
A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only α-helix 5 of domain I along with adjacent helices α-4 or α-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of α-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.
Role of DNA in the activation of the Cry1A insecticidal crystal protein from Bacillus thuringiensis
1998, Journal of Biological Chemistry
The Cry1A insecticidal crystal protein (protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA. Only the N-terminal toxic moiety of the protoxin was found to interact with the DNA. Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of protoxin, indicating that only a small region of the N-terminal toxic moiety interacts with the DNA. It is proposed that the DNA-protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward. It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the protoxin. Activation of the protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes. Larval trypsin initially converts the 20-kilobase pair DNA-protoxin complex to a 20-kilobase pair DNA-toxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.
Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells
1998, Biochimica et Biophysica Acta - Biomembranes
The size and ionic selectivity of the pores formed by the insecticidal crystal protein Cry1C from Bacillus thuringiensis in the plasma membrane of Sf9 cells, an established cell line derived from the fall armyworm Spodoptera frugiperda, were analyzed with a video imaging technique. Changes in the permeability of the membrane were estimated from the rate of osmotic swelling of the cells. In the presence of Cry1C, which is toxic to Sf9 cells, the permeability of the cell membrane to KCl and glucose increased in a dose-dependent manner. In contrast, Cry1Aa, Cry1Ab and Cry1Ac, toxins to which Sf9 cells are not susceptible, had no detectable effect. Pores formed by Cry1C allowed the diffusion of sucrose, but were impermeable to the trisaccharide raffinose. On the basis of the hydrodynamic radii of these substances, the diameter of the pores was estimated to be 1.0–1.2 nm. In the presence of salts, the rate of swelling of cells exposed to Cry1C was about equally influenced by the size of the anion as by that of the cation, indicating that the ionic selectivity of the pores is low.
Function and role of ATP-binding cassette transporters as receptors for 3D-cry toxins
2019, Toxins

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Quantification of the effect of Bacillus thuringiensis toxins on short-circuit current in the midgut of Bombyx mori

Abstract

Insect Biochem.

Gene

Comp. Biochem. Physiol.

Biochim. biophys. Acta

Comp. Biochem. Physiol.

Meth. Enzym.

FEBS Lett.

J. molec. Biol.

J. biol. Chem.

Biochem. biophys. Res. Commun.

J. biol. Chem.

Biochim. biophys. Acta

Biochem. biophys. Res. Commun.

J. biol. Chem.

Biochem. Physiol.

Conformational analysis of δ-endotoxins of Bacillus thuringiensis

Biophys. J.