Recombinant expression, purification, and characterization of scorpion toxin BmαTX14

Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.     

SAXS study of the snake toxin α-crotamine

-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (R _g=13.7 Å, S=3,000 Ų, V=9,200 ų and D _max=40 Å) were derived from SAXS curves obtained from a solution of this protein at pH=4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.     

A conserved tetrameric interaction of cry toxin helix α3 suggests a functional role for toxin oligomerization

Crystal (Cry) toxins are widely used for insect control, but their mechanism of toxicity is still uncertain. These toxins can form lytic pores in vitro, and water soluble tetrameric pre-pore intermediates have been reported. Even the precise oligomeric state of the toxin in membranes, trimeric or tetrameric, is still a debated issue. Based on previous reports, we have assumed that interactions between toxin monomers in solution are at least partly mediated by domain I, and we have analyzed in silico the homo-oligomerization tendencies of the domain I α-helices individually. Using many homologous sequences for each α-helix, our strategy allows selection of evolutionarily conserved interactions. These interactions appeared only in helices α3 and α5, but only α3 produced a suitably oriented or α-helical sample in lipid bilayers, forming homotetramers in C14-betaine, and allowing determination of its rotational orientation in lipid bilayers using site-specific infrared dichroism (SSID). The determined orientation in the tetrameric model is in agreement with only one of the evolutionarily conserved models. In addition mutation R99E, which was found to inhibit oligomerization experimentally, greatly destabilized the tetramer in molecular dynamic simulations. In this model, helix 3 is able to form inter-monomer interactions without significant rearrangements of domain I, which is compatible with the available crystal structure of Cry toxins in solution. The model presented here at least partially explains the reported tetrameric oligomerization of Cry toxins in solution and the inhibition of this oligomerization by a synthetic α3 peptide.     

Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard.     

Amino acid sequence of toxin VII,A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[¹⁴C] car?ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.     

The ζ toxin induces a set of protective responses and dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε(2)) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20-30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1-5×10(-5)). Early after induction ζ toxin alters the expression of ～78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K(+). We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε(2) antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (～10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε(2) antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.     

Determination of T-2 toxin,HT-2 toxin,and three other type A trichothecenes in layer feed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)—comparison of two sample preparation methods

A sensitive method for the simultaneous determination of T-2 toxin, HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol in layer feed using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry in the positive ionization mode (LC-ESI-MS/MS) is described. Two fast and easy clean-up methods—with BondElut Mycotoxin and MycoSep 227 columns, respectively—were tested. The separation of the toxins was conducted on a Pursuit XRs Ultra 2.8 HPLC column using 0.13 mM ammonium acetate as eluent A and methanol as eluent B. Detection of the mycotoxins was carried out in the multiple reaction monitoring (MRM) mode using ammonium adducts as precursor ions. Quantification of all analytes was performed with d₃-T-2 toxin as an internal standard. The clean-up method with MycoSep 227 columns gave slightly better results for layer feed compared to the method using BondElut Mycotoxin columns (MycoSep 227: recovery between 50 and 63 %, BondElut Mycotoxin: recovery between 32 and 67 %) and was therefore chosen as the final method. The limits of detection ranged between 0.9 and 7.5 ng/g depending on the mycotoxin. The method was developed for the analysis of layer feed used at carry-over experiments with T-2 toxin in laying hens. For carry-over experiments, it is necessary that the method includes not only T-2 toxin but also the potential metabolites in animal tissues HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol which could naturally occur in cereals used as feed stuff as well.     

Pasteurella multocida toxin activates Gβγ dimers of heterotrimeric G proteins

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G_q and G_12/13. PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCβ via Gα_q. Recent studies indicate that PMT additionally activates Gα_i to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP₃) as indicated by the recruitment of a PIP₃-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gα_q incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling.     

Cross-reactivity of monoclonal antibodies againstClostridium perfringens Θ toxin with streptolysin O

Six monoclonal antibodies to toxin ofClostridium perfringens were characterized. Four antibodies, 1C3, 2D4, 1B9, and 3F11, were nonneutralizing for toxin and were non-cross-reacting with streptolysin O (SLO). The other two antibodies, 3H10 and 2C5, were cross-binding and cross-neutralizing with SLO. Neutralizing activity of 3H10 was higher than that of 2C5 on the basis of the binding activity with toxin and SLO. Both antibodies could inhibit hemolysis even after binding of the toxins to sheep red blood cells and inhibited cardiotoxicity of the toxins in cultured heart cells.     

Role of phages in the pathogenesis of Burkholderia,or ‘Where are the toxin genes in Burkholderia phages?’

Most bacteria of the genus Burkholderia are soil- and rhizosphere-associated, and rhizosphere associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens, and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens; however, the data do not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a 'versaphile' such that selection is occurring in several niches, including as a pathogen and in the context of environmental survival.     

The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways

Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell?functions, but also disable macrophages and suppress inflammatory responses.     

Pollen-specific expression of theArabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genespromoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genes

We have characterized the promoter specificity of theArabidopsis thaliana α₁-tubulin (α ₁-tub) gene by studying expression patterns of gene fusions between the 2.2 kbp 5′ upstream region of theα ₁-tub gene and each of three different reporters: chloramphenical acetyltransferase, β-glucuronidase or the diphtheria toxin chain A gene. Analysis of transgenic tobacco andArabidopsis plants carrying the transgene showed that the chloramphenicol acetyltransferase and β-glucuronidase activities were not detected in any vegetative or reproductive organs except mature pollen. Transgenic tobacco plants carrying the diphtheria toxin chain A gene under the control of theα ₁-tub promoter were of normal phenotype but seed fertility was drastically reduced. Furthermore, the transgene could not be transmitted to the next generation through pollen, supporting the observation that theα ₁-tub promoter is active only in pollen. It was observed that the promoter activity was most active in mature pollen and decreased significantly duringin vitro pollen germination, indicating that the promoter is inactive or subdued in germinating pollen. The promoter activity was not affected by various plant growth hormones during pollen maturation.     

Nucellar callus of ‘Femminello’ lemon,selected for tolerance toPhoma tracheiphila toxin,shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., Femminello lemon) are inherently sensitive while others (e.g., Tarocco orange) are tolerant. Cell lines derived from nucellar tissue of Femminello, Tarocco and a cell line selected for tolerance to the fungal toxin (Femminello-S) were used to study host-pathogen interaction. Our results showed that calli or conditioned media of Tarocco and Femminello-S inhibited the size of co-cultivated fungal colonies when compared to Femminello. In addition, conditioned medium of Tarocco as well as FemminelloS, but not Femminello, promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in Femminello-S extracellular extracts as compared to Femminello. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of ¹²⁵I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α1₂β1_γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC₅₀ = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH₄C₁ cell line also showed the presence of activity close to that of the natural toxin (IC₅₀ 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain ¹⁵N-labeled analog of the neurotoxin.     

Characterization of variant diphtheria toxin–interleukin-3 fusion protein,DTIL3K116W,for phase I clinical trials

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin–interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at ?80 °C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography–HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296 EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC₅₀ of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC₅₀ of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at ?80 °C. Further, the drug was stable at 4 and 25 °C in the plastic syringe and administration tubing for 48 h.     

Electrophysiological response of chicken’s jejunal epithelium to increasing levels of T-2 toxin

The present investigations were conducted to test the effects of T-2 toxin on electrophysiological variables of jejunal epithelium of chicken. Jejunal segments of broilers were monitored in Ussing chambers in the presence of T-2 toxin at the levels of 0 (negative control), 0 (methanol/vehicle control), 0.1, 1, 5, and 10 μg/ml of buffer. T-2 toxin did not affect basal values of short circuit current (I_sc), transmural potential difference, or tissue conductivity in the jejunal epithelium. T-2 toxin also did not statistically affect glucose-induced electrophysiological variables during the first 3 min of glucose induction. Compared to the vehicle control, the ouabain-sensitive I_sc was negatively affected (P?=?0.008) only under 5 μg of T-2 toxin/ml. Increasing levels of T-2 toxin negatively affected the ouabain-sensitive I_sc in a cubic (P?=?0.007) fashion. These data indicate that acute exposure to moderate levels of T-2 toxin may progressively impair the cation gradient across the jejunal epithelium.     

Characterization and comparative analysis of toxin–antitoxin systems in Acetobacter pasteurianus

Journal of Industrial Microbiology & Biotechnology - Bacterial toxin–antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three...