David J. Ellar

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

A 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ‐endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N‐terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3‐fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with different insect specificity

The mechanism of action of Bacillus thuringiensis insecticidal δ-endotoxins has long been the subject of controversy. As our working hypothesis we propose a two-step model in which, after binding a specific plasma membrane receptor, the action of all the δ-endotoxins studied here is to generate small pores in the plasma membrane, either directly by inserting into the membrane, or indirectly by perturbing resident plasma membrane molecules. The creation of these pores will lead to colloid-osmotic lysis, i.e., an equilibration of ions through the pore resulting in a net inflow of ions, an accompanying influx of water, cell swelling and eventual lysis. Our observations that cell swelling precedes lysis, that small molecules leak out of the cell before large ones, that osmotic protectants inhibit or delay cytolysis, and that the toxin-induced pore of 0.5–1.0 nm radius will allow equilibration of ions but not leakage of cytoplasmic macromolecules, are in full agreement with the predictions of this hypothesis. To explain the specificity of the δ-endotoxin-induced lytic pore formation, we propose that prior interaction between the toxin and cell-specific plasma membrane recpetors is necessary before these toxins can insert into, or interact with, the membrane.

Mechanism of action of Bacillus thuringiensis var israelensis insecticidal δ-endotoxin

Bacillus thuringiensis var israelensisδ‐endotoxin protein active against mosquitoes was inactivated by prior incubation with lipids extracted from Aedes albopictus cells. Experiments with lipid dispersions and multilamellar liposomes showed that the toxin binds to phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine provided these lipids contain unsaturated fatty acids. Phosphatidyl serine binds toxin less efficiently and phosphatidyl inositol, cardiolipin, cerebroside and cholesterol show no affinity for the toxin. The results suggest an insecticidal mechanism in which interaction of toxin with specific plasma membrane lipids causes a detergent‐like rearrangement of the lipids, leading to disruption of membrane integrity and eventual cytolysis.

Comparative Toxicity of Bacillus thuringiensis var. israelensis Crystal Proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.

Surface plasmon resonance analysis at a supported lipid monolayer

Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.

Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1.

The bacterium Bacillus thuringiensis synthesizes toxins (δ‐endotoxins) that are highly specific for insects. Once ingested, the activated form of the toxin binds to a specific receptor(s) located on the midgut epithelial cells, inserts into the membrane causing the formation of leakage pores and eventual death of the susceptible insect larvae. Manduca sexta larvae are highly susceptible to Cry1Ac1, a toxin that is believed to bind M. sexta Aminopeptidase N, a glycoprotein located on the apical membrane. However, the binding data obtained to date only support the interaction of Cry1Ac1 with APN in vitro. To explore the in vivo role of APN, we have utilized the GAL4 enhancer trap technique to drive the expression of M. sexta APN in both midgut and mesodermal tissues of Cry1Ac1 insensitive Drosophila larvae. Transgenic Drosophila fed the toxin were now killed, demonstrating that APN can function as a receptor for Cry1Ac1 in vivo.

A cytolytic δ-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal δ‐endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation‐selective channels in the bilayers, permeable to K+ and Na+ but not to N‐methylglucamine or Cl−, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid‐osmotic lysis.

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (bbmvs) prepared from insect midguts. We observed that the sugar V-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CrylA (c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexta bbmvs, partially inhibited binding to Heliothis virescens bbmvs and had no apparent effect on binding to Pieris brassicae bbmvs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M . sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M . sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that V-acetyl galactosamine might be a component of a CrylA (c) toxin receptor of CF1 cells and of at least two of the insects tested.

Involvement of a possible chaperonin in the efficient expression of a cloned CryllA δ‐endotoxin gene in Bacillus thuringiensis

The Bacillus thuringiensis cryllAδ‐endotoxin gene is found as the third‐gene in a three‐gene operon, with a sporulation‐dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Lectin-like binding of Bacillus thuringiensis var, kurstaki lepidopteran-specific toxin is an initial step in insecticidal action

The two δ‐endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran‐specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N‐acetylgalactosamine and N‐acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N‐acetylgalactosamine, also inhibited toxicity. Since N‐acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N‐acetylgalactosamine residue.