Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing.

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 µg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

Canavalia ensiformis urease, Jaburetox and derived peptides form ion channels in planar lipid bilayers.

Ureases catalyze the hydrolysis of urea into NH3 and CO2. They are synthesized by plants, fungi and bacteria but not by animals. Ureases display biological activities unrelated to their enzymatic activity, i.e., platelet and neutrophil activation, fungus inhibition and insecticidal effect. Urease from Canavalia ensiformis (jack bean) is toxic to several hemipteran and coleopteran insects. Jaburetox is an insecticidal fragment derived from jack bean urease. Among other effects, Jaburetox has been shown to interact with lipid vesicles. In this work, the ion channel activity of C. ensiformis urease, Jaburetox and three deletion mutants of Jaburetox (one lacking the N-terminal region, one lacking the C-terminal region and one missing the central ß-hairpin) were tested on planar lipid bilayers. All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states whose conductance ranges were 7-18pS and 32-79pS, respectively. Urease and the N-terminal mutant of Jaburetox were more active at negative potentials, while the channels of the other peptides did not display voltage-dependence. This is the first direct demonstration of the capacity of C. ensiformis urease and Jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be a crucial step of their diverse biological activities, including host defense.

Global functional analyses of cellular responses to pore-forming toxins.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.

Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review.

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.

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles.

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.

Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation.

Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices alpha4 and alpha5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.

Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes.

Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

Effects of mutations within surface-exposed loops in the pore-forming domain of the Cry9Ca insecticidal toxin of Bacillus thuringiensis.

The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca.

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 degrees C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.

Helix alpha 4 of the Bacillus thuringiensis Cry1Aa toxin plays a critical role in the postbinding steps of pore formation.

Helix alpha 4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix alpha 4 amino acid residues have previously been shown to cause substantial reductions in the protein's pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix alpha 4 is involved mainly in the postbinding steps of pore formation.

Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis.

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action.

The influence of membrane bilayer thickness on KcsA channel activity.

Atomic resolution structures have provided significant insight into the gating and permeation mechanisms of various ion channels, including potassium channels. However, ion channels may also be regulated by numerous factors, including the physiochemical properties of the membrane in which they are embedded. For example, the matching of the bilayer's hydrophobic region to the hydrophobic external surface of the ion channel is thought to minimize the energetic penalty needed to solvate hydrophobic residues or exposed lipid tails. To understand the molecular basis of such regulation by hydrophobic matching requires examining channels in the presence of the lipid membrane. Here we examine the role of hydrophobic matching in regulating the activity of the model potassium channel, KcsA. 86Rb+ influx assays and single-channel recordings indicate that the non-inactivating E71A KcsA channel is most active in thin bilayers (

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac.

After binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed.

Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa.

Helix alpha4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the alpha4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

Estimation of the radius of the pores formed by the Bacillus thuringiensis Cry1C delta-endotoxin in planar lipid bilayers.

Pore formation constitutes a key step in the mode of action of Bacillus thuringiensis delta-endotoxins and various activated Cry toxins have been shown to form ionic channels in receptor-free planar lipid bilayers at high concentrations. Multiple conductance levels have been observed with several toxins, suggesting that the channels result from the multimeric assembly of a variable number of toxin molecules. To test this possibility, the size of the channels formed by Cry1C was estimated with the non-electrolyte exclusion technique and polyethylene glycols of various molecular weights. In symmetrical 300 mM KCl solutions, Cry1C induced channel activity with 15 distinct conductance levels ranging from 21 to 246 pS and distributed in two main conductance populations. Both the smallest and largest conductance levels and the mean conductance values of both populations were systematically reduced in the presence of polyethylene glycols with hydrated radii of up to 1.05 nm, indicating that these solutes can penetrate the pores formed by the toxin. Larger polyethylene glycols had little effect on the conductance levels, indicating that they were excluded from the pores. Our results indicate that Cry1C forms clusters composed of a variable number of channels having a similar pore radius of between 1.0 and 1.3 nm and gating synchronously.

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles.

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg(2+), Ca(2+) and Ba(2+)) and anions (SO(4)(2-) and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.

Patch-clamp study of the apical membrane of the midgut of Manduca sexta larvae: direct demonstration of endogenous channels and effect of a Bacillus thuringiensis toxin.

The patch-clamp technique was applied to the apical membrane of epithelial midgut cells of a lepidoptera, Manduca sexta L. Access to the apical membrane, the main target site of Bacillus thuringiensis (Bt) toxins, was achieved by using freshly isolated larval midgut preparations mounted onto holding glass pipettes. The epithelial cells retained their functional integrity, as evidenced by the magnitude of intracellular potentials recorded with microelectrodes. With standard 32 mM K(+) solution in the bath and the patch-clamp pipette, endogenous channel activity was detected in about 50% of experiments, mainly in moulting larvae and larvae that had been kept at reduced temperature for at least two days prior to the experiments. In both cell-attached and inside-out patch-clamp configurations, different types of channel were observed, with conductances varying between about 5 and 50 pS and different conducting properties. Addition of trypsin-activated Cry1Ac Bt toxin in the patch-clamp pipette triggered, after a delay, large conductances of a few nanosiemens. This is the first study allowing exploration, in the intact midgut, of the properties of apical membrane channels and the direct interaction between the apical membrane of epithelial cells and pathogenic agents such as Bt toxins.