Individual Toxins Research Articles

Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry

The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Enhancing the Efficacy of Energy Psychology Psychotherapies by Neutralizing Individual Energy Toxins

Enhancing the Efficacy of Energy Psychology Psychotherapies by Neutralizing Individual Energy Toxins

The Aerolysin-Like Toxin Family of Cytolytic, Pore-Forming Toxins

Pore-forming toxins (PFTs) represent the largest known group of bacterial protein toxins to date. Membrane insertion and subsequent pore-formation occurs after initial binding to cell-surface receptor and oligomerization. Aerolysin, a toxin produced by the Gram-negative bacterium Aeromonas hydrophila and related species, belongs to the PFT group and shares a common mechanism of action involving  -barrel structures resulting from the assembly of  - hairpins from individual toxin monomers into a heptamer. Aerolysin is also the name given to structurally and mechanistically related toxins called the aerolysin-like toxin family. A universal characteristic of this toxin family involves the diverse life forms that synthesize these proteins throughout Nature. Examples include: 1) epsilon-toxin and septicum-alpha-toxin produced by anaerobic, Gram-positive Clostridium species; 2) enterolobin by the Brazilian tree Enterolobium contortisiliquum; 3) a mushroom toxin Laetiporus sulphureus lectin (LSL); 4) mosquitocidal toxins (Mtxs) from the Gram-positive bacteria Bacillus sphaericus and parasporine-2 from Bacillus thuringiensis; and 6) hydralysins from the tiny aquatic animal Chlorohydra viridis. The following review provides an overview of the different members within the aerolysin-like toxin family.

CRY4AA AND CRY4BA FROM BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS EXPRESSED IN INSECT CELLS BY RECOMBINANT BACULOVIRUSES ARE TOXIC TO AEDES AEGYPTI LARVAE

Bacillus thuringiensis subsp. israelensis (Bti) is highly toxic to mosquito larvae. Most of its toxicity relies on δ-endotoxins (Cry and Cyt) crystalline inclusions produced at the sporulation phase of growth. Obtention of individual Bti mosquitocidal toxins is crucial for the understanding of Bti´s potential activity against dipteran larvae. A Cry toxin expression system based on baculovirus was developed to produce two of the Cry toxins comprised in the crystals of Bti. Th e cry4Aa and cry4Ba genes from two Brazilian strains of Bacillus thuringiensis were amplified by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis of the cry4Aa and cry4Ba genes showed high identity to previous known cry genes. Both cry4Aa and cry4Ba genes were further cloned into transfer vectors for construction of recombinant baculoviruses. After isolation of the recombinant viruses, they were used to infect insect cells (BTITN5B1-4) that were analyzed by light microscopy at different times post infection. Putative crystals consisting of either Cry4Aa or Cry4Ba were observed in the cytoplasm of infected insect cells. RT-PCR was performed with mRNA from insect cell extracts (72 h.p.i.) in order to confirm the presence of the genes specific transcripts. Recombinant virus-infected insect extracts (120 h.p.i.) were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) showing the presence of polypeptide bands of around 128 and 130 kDa, corresponding, respectively to the sizes of the proteins Cry4Aa and Cry4Ba. Bioassays with virus-infected insect extracts were shown to be toxic to second instar Aedes aegypti larvae, confirming the usefulness of this expression system for the study of Cry proteins. DOI: http://dx.doi.org/10.17525/vrr.v18i1-2.70

Predicting function from sequence in a large multifunctional toxin family

Venoms contain active substances with highly specific physiological effects and are increasingly being used as sources of novel diagnostic, research and treatment tools for human disease. Experimental characterisation of individual toxin activities is a severe rate-limiting step in the discovery process, and in-silico tools which allow function to be predicted from sequence information are essential. Toxins are typically members of large multifunctional families of structurally similar proteins that can have different biological activities, and minor sequence divergence can have significant consequences. Thus, existing predictive tools tend to have low accuracy. We investigated a classification model based on physico-chemical attributes that can easily be calculated from amino-acid sequences, using over 250 (mostly novel) viperid phospholipase A2 toxins. We also clustered proteins by sequence profiles, and carried out in-vitro tests for four major activities on a selection of isolated novel toxins, or crude venoms known to contain them. The majority of detected activities were consistent with predictions, in contrast to poor performance of a number of tested existing predictive methods. Our results provide a framework for comparison of active sites among different functional sub-groups of toxins that will allow a more targeted approach for identification of potential drug leads in the future.

Temperature effects on kinetics of paralytic shellfish toxin elimination in Atlantic surfclams, Spisula solidissima

Surfclams, Spisula solidissima, pose a particular health risk for human consumption as they are characterized by accumulation of extremely high levels of toxins associated with paralytic shellfish poisoning (PSP), slow toxin elimination and an extremely high post-ingestive capacity for toxin bioconversion. Surfclam populations experience a wide range of temperatures along the NW Atlantic continental shelf, and are undergoing range contraction that has been attributed to global warming. In this study the influence of temperature (5, 12 and 21°C) on detoxification kinetics of individual PSP toxins in two tissue compartments of juvenile surfclams (∼35mm shell length) was determined under controlled laboratory conditions, over prolonged (2.4 months) depuration. Clams were toxified with a representative regional Gulf of Maine isolate of the dinoflagellate Alexandrium fundyense of known toxin profile, allowing tracking of changes in toxin composition and calculated toxicity in surfclam tissues. The visceral mass detoxified at all temperatures, although toxin loss rate increased with increasing temperature. In contrast, total toxin content and calculated toxicities in other tissues remained constant or even increased during depuration, suggesting a physiological or biochemical toxin-retention mechanism in this tissue pool and temperature-independent detoxification. In vivo toxin compositional changes in surfclam tissues found in this study provide evidence of specific toxin conversion pathways, involving both reductive and decarbamoylation pathways. We conclude that such toxin biotransformations, especially in non-visceral tissues, may introduce a discrepancy in describing kinetics of total toxicity (in saxitoxin equivalents [STXeq]) of S. solidissima over prolonged detoxification. Nevertheless, use of total toxicity values generated by routine regulatory monitoring based upon mouse bioassays or calculated from chemical analytical determination of molar toxin concentrations is adequate for first-order modeling of toxin kinetics in this species. Furthermore, the differential detoxification response of viscera and other tissues in relation to temperature emphasizes the need for two-compartment modeling to describe the fate of PSP toxins in this species. Finally, key parameters were identified that may prove useful in hindcasting the timing of toxic blooms or new toxin input in deep offshore waters where routine monitoring of toxic phytoplankton is impractical.

INCREASE IN LARVAL GUT PROTEOLYTIC ACTIVITIES AND Bti RESISTANCE IN THE DENGUE FEVER MOSQUITO

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.

Neutralization of B. anthracis toxins during ex vivo phagocytosis

Glycoconjugates (GCs) are recognized as stimulation and signaling agents, affecting cell adhesion, activation, and growth of living organisms. Among GC targets, macrophages are considered ideal since they play a central role in inflammation and immune responses against foreign agents. In this context, we studied the effects of highly selective GCs in neutralizing toxin factors produced by B. anthracis during phagocytosis using murine macrophages. The effects of GCs were studied under three conditions: A) prior to, B) during, and C) following exposure of macrophages to B. anthracis individual toxin (protective antigen [PA], edema factor [EF], lethal factor [LF] or toxin complexes (PA-EF-LF, PA-EF, and PA-LF). We employed ex vivo phagocytosis and post-phagocytosis analysis including direct microscopic observation of macrophage viability, and macrophage activation. Our results demonstrated that macrophages are more prone to adhere to GC-altered PA-EF-LF, PA-EF, and PA-LF toxin complexes. This adhesion results in a higher phagocytosis rate and toxin complex neutralization during phagocytosis. In addition, GCs enhance macrophage viability, activate macrophages, and stimulate nitric oxide (NO) production. The present study may be helpful in identifying GC ligands with toxin-neutralizing and/or immunomodulating properties. In addition, our study could suggest GCs as new targets for existing vaccines and the prospective development of vaccines and immunomodulators used to combat the effects of B. anthracis.

Disease facts: Mycotoxicosis

Mycotoxins are widespread contaminants of pig feed and bedding. A wide range of effects can occur as a result of individual or combined toxins. Food sourcing, handling and storage are key to minimising the presence of mould toxins and their effects.

Susceptibility of field populations of sugarcane borer from non‐Bt and Bt maize plants to five individual Cry toxins

Abstract Sugarcane borer, Diatraea saccharalis (F.), is a major target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the US mid‐south region. Resistance development in target pest populations is a major threat to the sustainable use of Bt crops. In our field trials in 2009, a significant number of live borers and plant injury from D. saccharalis were observed in an experimental SmartStax™ maize line. The objective of this study was to assess the relative susceptibility of two field populations of D. saccharalis collected from non‐Bt and Bt maize plants containing SmartStax™ traits to five individual Cry proteins. The five Bt proteins included two proteins (Cry1A.105 and Cry2Ab2) that were expressed in SmartStax™ maize plants and three other common Bt proteins (Cry1Aa, Cry1Ab and Cry1Ac) that were not produced in SmartStax™. Larval mortality and growth inhibition on Bt diet of the fourth generation after field collections were evaluated 7 days after release of neonates on the diet surface. The laboratory bioassays showed that 50% lethal concentration (LC50) values for Cry1A.105 and Cry2Ab2 for the population originated from Bt plants were 3.55‐ and 1.34‐fold greater, respectively, than those of the population collected from non‐Bt plants. In contrast, relative to the population from non‐Bt plants, the LC50 of the population sampled from Bt plants were 3.85‐, 2.5‐ and 1.64‐fold more sensitive to Cry1Aa, Cry1Ab and Cry1Ac, respectively. The results did not provide clear evidence to conclude that the observed field survival of D. saccharalis on Bt plants was associated with increased levels of resistance.

Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics

Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codon-optimized for expression in Escherichia coli and encoded a 26kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (~28kDa) and Tsst-1 (~22kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.

Uptake and release of paralytic shellfish toxins by the clam Ruditapes decussatus exposed to Gymnodinium catenatum and subsequent depuration

A laboratory experiment was performed with the clam Ruditapes decussatus, fed with the toxic dinoflagellate Gymnodinium catenatum and the non-toxic algae Isochrysis galbana (14 days) and subsequently only with I. galbana (15 days). Individual paralytic shellfish toxins were determined by LC-FLD in G. catenatum cells, whole clam tissues, and particulate organic matter (POM) produced by clams. The toxins dcSTX and dcGTX2 + 3 in the algae were less abundant than C1 + 2 and B1, but were predominant in clams during both the exposure and depuration phases. The toxin dcNEO was only detected in clams during a short period, indicating conversion from other compounds. The toxin composition of the POM indicated the export of dcSTX as faeces or pseudo-faeces along the entire experiment (2.5–14 nmol mg−1), B1 was present in a short period of the exposure and C1 + 2 and dcGTX2 + 3 absent. A mass balance calculation indicated that approximately 95% of C1 + 2 and 85% of B1 supplied to the clams were converted into other toxins or lost in solution. Conversely, the net gain of 512, 61 and 31 nmol for dcSTX, dcGTX2 + 3 and dcNEO, respectively, suggests the conversion from other assimilated compounds by clams during exposure and depuration phases.

Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded.

Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli.

Studies on apoptotic changes in combined toxicity of citrinin and endosulfan in pregnant Wistar rats and their fetuses

Background:Citrinin (mycotoxin) and endosulfan (pesticide) both environmental contaminants easily enter the food chain and are caoomon causes of various toxicities.Materials and Methods:In the present investigation, citrinin (CIT) (10 mg/kg feed) and endosulfan (1 mg/kg body weight) were administered orally alone and in combination to pregnant Wistar rats from gestational day 6 to 20 to study their effect to cause apoptosis in the pregnant Wistar rats and their fetuses. Apoptosis was assessed in dams by agarose gel electrophoresis, flow cytometry and electron microscopy, while in the fetuses it was assessed by flow cytometry only.Result:Citrinin and endosulfan in the combination group caused apoptosis in an additive manner as there was increased number of apoptotic cells as compared to the individual toxin and control groups. The fetuses also showed increased number of apoptotic cells in the combination groups, which also indicated that both the toxins crossed the placental barrier.Conclusion:So it was concluded that apoptosis played a significant role in the pathogenesis of endosulfan and citrinin toxicity.

Effects by anthrax toxins on hematopoiesis: A key role for cytokines as mediators

An understanding of anthrax toxins on the emerging immune system and blood production are significant to medicine. This study examined the effects of anthrax toxin on hematopoiesis and determined roles for cytokines. Anthrax holotoxin toxin is three components: protective antigen (PA) binds to the target cell and mediates the entry of lethal factor (LF) and edema factor (EF). Anthrax toxin dramatically inhibits signaling in immune cells. We first identified the cell subsets that interacted with the protective antigen (PA) and then studied the effects on hematopoietic progenitors in clonogenic assays: granulocytic–monocytic (CFU-GM) and late erythroid (CFU-E). Multi-color immunofluorescence with FITC-PA indicated its interaction with early and late myeloid cells. Clonogenic assays, in the presence or absence of holotoxin and individual toxin proteins resulted in significant suppression by hologenic toxic alone, despite the presence of growth-promoting cytokines. Antibodies to anthrax receptor (ATR1) reversed the suppressive effects, indicating specificity. Monomeric proteins showed different effects on myeloid and erythroid progenitors. Suppression was not due to cell death, based on undetectable active caspase 3. Cytokine array analyses with supernatants from toxin-stimulated stroma showed an increase in the hematopoietic suppressor, MIP-1α. This finding, in addition to our previous studies, showing an increase in IL-10, suggested indirect roles for cytokines in toxin-mediated hematopoietic suppression. The chemokine, SDF-1α was increased. Since SDF-1 is involved in the mobilization of hematopoietic cells, it is likely that anthrax holotoxin could induce cell exit from BM. In summary, anthrax holotoxin, but not individual toxins, exerted hematopoietic effects on myeloid and erythroid progenitors via specific receptor, partly through the induction of cytokines.

How best to improve survival in hemodialysis patients: solute clearance or volume control?

How much dialysis is required for optimal well-being and long-term survival? The Frequent Hemodialysis Network completed two prospective trials examining the effect of more frequent, short hours and, now, of nocturnal hemodialysis compared with standard thrice-weekly treatments. Whereas the short-hours trial reported benefits with more frequent dialysis, somewhat paradoxically, nocturnal hemodialysis had fewer advantages. However, this trial was probably underpowered, and confounded by the beneficial effects of home hemodialysis in the control group.

Immune Response Towards Snake Venoms

The immune response involves a complex repertoire of innate and adaptive responses to foreign agents in the organism. The present review focuses on the immune response to snake venoms, including those occurring in snakebite accidental envenomation, experimental vaccination and animal hyperimmunization for snake antivenom production. The following aspects are considered: (a) the structural characteristics of snake toxins and their relationship to immunogenicity, (b) the effects that factors such as administration route, venom dose, type of adjuvant, and individual and species characteristics of the immunized animal have on the immune response, (c) the initial venom-induced inflammatory response, (d) the process by which specific antibodies towards individual toxins are produced, and (e) the techniques currently used to evaluate the antibody response. Understanding the immune response to snake venoms is highly relevant for improving antivenom production and for gaining a more complete view of snakebite envenoming.