Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria.

Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.     

Theta-toxin of Clostridium perfringens. I. Purification and some properties.

Theta-Toxin, an oxygen-labile hemolysin produced by Clostridium perfringens, was purified 3300 fold from culture filtrate by successive chromatography on DEAE-Sephadex A-50 and Sephadex G-150. The purified toxin gave two distinct bands in disc electrophoresis, while the same material, after mild reduction with dithiothreitol, yielded a single band, indicating that the purified theta-toxin contained, as well as a reduced, active form, an oxidized, inactive form of toxin. These two forms of the toxin had a similar, if not identical molecular size. The purified preparation gave a single band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis and formed a single precipitin line with National Standard gas gangrene (C. perfringens) antitoxin. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of theta-toxin was estimated to be 51 000, the value being in exact accordance with that obtained by amino acid analysis. The amino acid composition of theta-toxin was very close to that of cereolysin, an oxygen-labile hemolysin produced by Bacillus cereus. The amino-terminal residue of theta-toxin was lysine as determined by the Dansyl method.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa.

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus

A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.     

Functional domains of Bacillus thuringiensis insecticidal crystal proteins. Refinement of Heliothis virescens and Trichoplusia ni specificity domains on CryIA(c)

Insecticidal crystal proteins (delta-endotoxins), CryIA(a) and CryIA(c), from Bacillus thuringiensis are 82% homologous. Despite this homology, CryIA(c) was determined to have 10-fold more insecticidal activity toward Heliothis virescens and Trichoplusia ni than CryIA(a). Reciprocal recombinations between these two genes were performed by the homolog-scanning technique. The resultant mutants had different segments of their primary sequences exchanged. Bioassays with toxin proteins from these mutants revealed that amino acids 335-450 on CryIA(c) are associated with the activity against T. ni, whereas amino acids 335-615 on the same toxin are required to exchange full H. virescens specificity. One chimeric protein toxin, involving residues 450-612 from CryIA(c), demonstrated 30 times more activity against H. virescens than the native parental toxin, indicating that this region plays an important role in H. virescens specificity. The structural integrity of mutant toxin proteins was assessed by treatment with bovine trypsin. All actively toxic proteins formed a 65-kDA trypsin-resistant active toxic core, similar to the parental CryIA(c) toxin, indicating that toxin protein structure was not altered significantly. Contrarily, certain inactive mutant proteins were susceptible to complete protease hydrolysis, indicating that their lack of toxicity may have been due to structural alterations.     

Yeast killer toxin: purification and characterisation of the protein toxin from Saccharomyces cerevisiae.

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl-controlled-pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11,470-Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non-killer showed the presence of the toxin protein only in the killer-derived material. The activity of the toxin was most stable between pH 4.2 and 4.6. At 30 degrees C toxin from a superkiller strain was more stable than that from a normal killer.     

T-2 Toxin Production by Fusarium tricinctum on Solid Substrate

A method has been developed to produce and purify gram quantities of T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3alpha-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.     

Detection of neutral sugars in purified type G botulinum progenitor toxin and the effects of some glycolytic enzymes on its molecular dissociation and oral toxicity

Arabinose and galactose were detected in purified type G botulinum toxin (Mr about 500,000) of Clostridium argentinense. The i.p. LD50/mg N of type G progenitor toxin was one-tenth, but the oral LD50/mg N twice that of type A-L toxin. The lysozyme-, endo-beta-galactosidase-, and N-glucanase-treated toxins each had a molecular mass of about 300,000. The oral toxicity of the endo-beta-galactosidase or N-glucanase-treated toxin was one-fifth that of untreated progenitor toxin. On DEAE-Sephadex chromatography, the N-glucanase-treated toxin dissociated into two fractions, nontoxic and toxic. SDS-PAGE of the toxic fraction showed a single band with a Mr of about 150,000, and after dithiothreitol treatment, two bands with Mr of 100,000 and 50,000.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Biochemical and physiological characterization of a new Na+-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12 Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na⁺-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.     

A new polypeptide toxin from the nematocyst venom of an Okinawan sea anemone Phyllodiscus semoni (Japanese name "unbachi-isoginchaku")

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).     

Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.