Barbara H. Knowles

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 Insecticidal δ-Endotoxins

Publisher Summary This chapter provides an overview of the biology of Bacillus thuringiensis ( Bt ). The chapter examines how the recently solved X-ray crystal structure of one Bt toxin allows to model the mode of action of a whole family of related protein toxins, and how a knowledge of the physiology of the insect targets of Bt toxins is needed for a better understanding of the toxic mechanism. Bacillus thuringiensis ( Bt ), a family of bacteria which make insecticidal proteins, accounts for 90–95% of the insect biocontrol market. This chapter illustrates that Bt is the name given to a family of bacteria found throughout the world. Paradoxically, CryIIIA, the only Bt toxin whose three-dimensional structure has been solved, kills very small insects, and its insolubility at neutral pH makes it difficult to assay in vitro . The crystal structure of CryIIIA permits site-directed mutagenesis and segment swapping experiments to be designed in a rational manner in order to identify the functional domains of the toxins. This chapter explores one attractive prospect that can be the replacement of the specificity domain with a binding domain directed to a target of choice, opening up the possibility of “designer” pesticides or a new class of immunotoxins. Characterization of toxin receptors and investigation of the mechanism of toxin synergism may assist in strategies for resistance management, a vital concern if Bt is to maintain its important position as the most extensively used biological insecticide.

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.

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.

A Broad-Spectrum Cytolytic Toxin from Bacillus thuringiensis var. kyushuensis

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23—22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cationselective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73—E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.

Mechanism of action of Bacillus thuringiensis israelensis parasporal body.

The Bacillus thuringiensis subsp. israelensis (B.t.i.) parasporal body is a gut poison, and the midgut epithelium of affected larvae is considered to be its initial site of action (de Barjac 1978). In B.t.i. -treated mosquito larvae, midgut epithelial cells swell and burst, causing severe damage to the gut wall (de Barjac 1978; Charles and de Barjac 1983; Lahkim-Tsror et al. 1983). The general characteristics of poisoning of B.t.i. -treated mosquito larvae (4 µg/ml) are cessation of feeding within one hour, reduced activity by two hours, extreme sluggishness by four hours, and general paralysis by six hours. Singh, Schouest, and Gill (1986) showed that the effect on the posterior midgut ultrastructure was detected as early as one hour, when the epithelial striated border was damaged. This was followed by swelling of epithelial organelles. Midgut circular and longitudinal muscles were also damaged, as indicated by their swelling and separation from the basement lamina. Six hours after B.t.i. treatment the midgut wall was severely ruptured, with the peritrophic membrane and basement lamina broken. Of these lesions, disruption of microvilli and epithelial swelling in the midgut coincide with cessation of feeding. The sluggish behavior and paralysis of insects only occurred in advanced stages of parasporal body poisoning.

Nonspecific ionic effects on the cytolytic and hemolytic properties ofBacillus thuringiensis δ-endotoxins

We investigated the in vitro effects of ions, carbohydrates, lectins, and charged compounds on the cytolytic and hemolytic action of the purified δ-endotoxin ofBacillus thuringiensis var.darmstadiensis 73-E10-2, and other δ-endotoxins. Cytotoxicity was inhibited by preincubating the toxin with N-acetylneuraminic, glucuronic, galacturonic, and N-acetylglutamic acids and ATP. Lectins were unable to inhibit toxicity. Pretreatment of sheep erythrocytes with neuraminidase enhanced hemolysis. These results suggested a nonspecific inhibition of cytotoxicity based on the presence of a negative charge. Supraphysiological concentrations of divalent cations such as Ca2+, Mg2+, and Zn2+ in the medium yielded a reduced toxicity, whereas EDTA and EGTA enhanced cytotoxicity.