Target cell specificity of the Pasteurella haemolytica leukotoxin is unaffected by the nature of the fatty-acyl group used to activate the toxin in vitro

The leukotoxin (LktA) of Pasteurella haemolytica is active only against cells of ruminant origin. It is synthesised as an inactive protoxin encoded by the lktA gene and post-translationally modified to the active toxin by the product of the lktC gene. The LktA and LktC proteins were expressed separately in Escherichia coli and partially purified. Active LktA was produced in vitro in the presence of LktC and acyl-acyl carrier protein (ACP) charged separately in vitro with a fatty-acyl group. The toxic activity and target cell specificity of LktA and adenylate cyclase toxin (CyaA), a toxin active against a wide variety of mammalian cells, were investigated after activation with ACP charged with different fatty acids. Palmitoyl-ACP produced the most active toxin in both cases and, although other fatty acids were also effective, the fatty acid preference was the same for the in vitro activation of both toxins. Activated LktA remained ruminant cell-specific whichever acyl group was used to acylate the A protoxin.     

Toxic Trypsin Digest Fragment from the Bacillus thuringiensis Parasporal Protein

Enzymatic digestion in vitro of the Bacillus thuringiensis protoxin presumably releases and activates the toxin in a manner analogous to that which occurs when a B. thuringiensis sporulated fermentation preparation passes through the midgut of a lepidopteran larva. Therefore, a sporulated culture of B. thuringiensis subsp. kurstaki (serotype 3a3b) HD-263 was treated with trypsin to release an activated toxin soluble in bicarbonate buffer. A 63-kilodalton protein, toxic to cabbage looper larvae (Trichoplusia ni) and to lepidopteran cells in culture, was purified to homogeneity from this trypsin digest. The larvicide, a glycoprotein containing 5% carbohydrate (wt/wt), was purified from the soluble B. thuringiensis trypsin digest by using ammonium sulfate precipitation, anion-exchange chromatography, and hydrophobic-interaction chromatography. Its amino acid composition was high in nonpolar residues and unusually low in lysine and histidine. The isoelectric point was 6.5, and the amino acid on the N terminus was identified as isoleucine. The toxin was only slightly soluble in aqueous buffers unless the chaotropic agent potassium thiocyanate was added. Partial characterization of the toxin indicated that it corresponds well with reported sequences deduced from cloned genes.     

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg¹⁵¹? Ser¹⁵² bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

     

Mapping and characterization of the entomocidal domain of the Bacillus thuringiensis CrylA(b) protoxin

The amino acid sequences necessary for entomocidal activity of the CryIA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that amino acid residues C-terminal to Ala607 are not required for insecticidal activity and that activation by midgut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the highly conserved β-strand, were prone to proteolytic degradation, explaining their lack of toxicity. Apparently, this β-strand is essential for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 28 codons, resulting in a translation product extending from amino acid 29 to 607. This protein appeared to be toxic not only to susceptible insect larvae such as Manduca sexta and Heliothis virescens, but also to Escherichia coli cells. An additional mutant, encoding only amino acid residues 29–429, encompassing the complete putative pore forming domain, but lacking a large part of the receptor-binding domain, was similarly toxic to E. coli cells. This suggests a role for the N-terminal 28 amino acids in rendering the toxin inactive in Bacillus thuringiensis, and indicates that the cytolytic potential of the pore forming domain is only realized after proteolytic removal of these residues by proteases in the insect gut. In line with this hypothesis are results obtained with a mutant protein in which Arg28 at the cleavage site was replaced by Asp. This substitution prevented the protein from being cleaved by trypsin in vitro, and reduced its toxicity to M. sexta larvae.     

Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA

The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K₅₆₄ (KI) and K₆₉₀ (KII) that are fatty acylated. Each domain required 15–30 amino acids for basal recognition and 50–80 amino acids for wild-type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca²⁺ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAll domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.     

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.     

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.     

Electrical hypothesis of toxicity of the Cry toxins for mosquito larvae

Many electrical properties of insect larval guts have been studied, but their importance for toxicity of the Cry-type toxins has never been reported in the literature. In the present work, we observed potential-dependent permeabilization of plasma membrane by several polycationic peptides derived from the Cry11Bb protoxin. The peptide BTM-P1d, all D-type amino acid analogue of the earlier reported peptide BTM-P1, demonstrated high membrane-permeabilizing activity in experiments with isolated rat liver mitochondria, RBC (red blood cells) and mitochondria in homogenates of Aedes aegypti larval guts. Two larger peptides, BTM-P2 and BTM-P3, as well as the Cry11Bb protoxin treated with the protease extract of mosquito larval guts showed similar effects. Only protease-resistant BTM-P1d, in comparison with other peptides, displayed A. aegypti larval toxicity. Taking into account the potential-dependent mechanism of membrane permeabilization by studied fragments of the Cry11Bb protoxin and the literature data related to the distribution of membrane and transepithelial potentials in the A. aegypti larval midgut, we suggest an electrical hypothesis of toxicity of the Cry toxins for mosquito larvae. According to this hypothesis, the electrical field distribution is one of the factors determining the midgut region most susceptible for insertion of activated toxins into the plasma membrane to form pores. In addition, potential-dependent penetration of short active toxin fragments into the epithelial cells could induce permeabilization of mitochondria and subsequent apoptosis or necrosis.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Variability in the cadherin gene in an Ostrinia nubilalis strain selected for Cry1Ab resistance

Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701 bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect.     

Functional Dissection of the Clostridium botulinum Type B Hemagglutinin Complex: Identification of the Carbohydrate and E-Cadherin Binding Sites

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.     

Expression and Crystallization of an N-Terminally Activated Form of the Bacillus thuringiensis Cry1Ca Toxin

When the active form of the Bacillus thuringiensis delta-endotoxin Cry1Ca was expressed in E. coli severe growth retardation was observed. The absence of a short peptide from the N-terminus of the protoxin was responsible for this effect. The introduction of a mutation at an amino acid previously reported as being involved in the initial stages of pore formation within the natural insect target partially abolished the growth retardation effect. We suggest that removal of the N-terminal peptide is a necessary step in toxin activation, the presence of this peptide preventing proper interaction of the toxin with the target membrane. Expression of the truncated toxin in Bacillus thuringiensis also prevented the formation of Cry1Ca crystals. Received: 7 March 2001 / Accepted: 12 April 2001     

Different Mechanisms of Resistance to Bacillus thuringiensis Toxins in the Indianmeal Moth

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of ¹²⁵I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688^s) and two resistant (198^r and Dpl^r) to Bacillus thuringiensis. Toxicological studies showed that the 198^r colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl^r colony was 4-fold more resistant to protoxin versus toxin. Binding results with ¹²⁵I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl^r colony showed a drastic reduction in binding affinity (60-fold higher K_d), although they had similar concentrations of binding sites. Insects from the 198^r colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K_d and ca. three-fold-lower R_t compared with the 688^s colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198^r colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.     

Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin.

The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.     

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

Cloning and Expression of the Insecticidal Crystal Protein Gene <Emphasis Type="Italic">Cry</Emphasis>1Ca9 of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> G10-01A from Taiwan Granaries

A new cry gene (cry1Ca9) was cloned and sequenced from a Bacillus thuringiensis isolate native to Taiwan (G10-01A). The cry1C-type gene, designated cry1Ca9, consisted of an open reading frame of 3,567 bp, encoding a protein of 1,189 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa. The gene sequence was compared against the GenBank nucleotide sequence data base. It was found that the cry1Ca9 gene coded for a 134.7-kDa protoxin which had greater than 99.8% homology with the previously reported cry1Ca1 gene, as only three mismatches were found between the two amino acid sequences. When the Cry1Ca9 toxin was expressed in a crystal-negative strain of B. thuringiensis (cryB^-), elliptical crystals were produced. Cell extracts from this recombinant strain appear to have high insecticidal activity against lepidopteran larvae (Plutella xylostella).Received: 23 September 2002 / Accepted: 6 December 2002     

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.     

A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum

In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.