Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.     

N‐terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

Mannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase‐9‐dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N‐terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N‐terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagicEscherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N‐terminal region in addressing certain RTX toxins to mitochondria.     

赤潮藻毒素生物合成研究进展

合成毒素是赤潮藻类的一个常见特征,已知能够产生毒素的微藻有70多种。作为次级代谢产物,藻毒素的产生可能是一种压制或清除其它藻类竞争者的一种反应,在群落演替、种间竞争中发挥重要作用。目前,人们对藻毒素生物合成机理依然知之甚少,相关基因的研究仍无明显突破。利用环境因子诱导毒素生成变化进而分离差异表达基因或者比较不同产毒藻株间基因表达的差异,从中克隆藻毒素生物合成基因似乎是一种极具潜力的研究方向。     

Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Toxins-antitoxins: diversity,evolution and function

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.     

Toxins-antitoxins: diversity, evolution and function

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.     

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.     

A comparative study of ricin and diphtheria toxin-antibody-conjugate kinetics on protein synthesis inactivation

Kinetic data on toxin and antibody-toxin-conjugate inactivation of protein synthesis have been used to assess the variables which affect the transport of these toxins into the cytosol compartment. First-order inactivation rate constants of protein synthesis (ki) are compared under conditions of known receptor occupancy. The effect of inclusion of toxin B chains, both homologous and heterologous, in antibody-toxin conjugates is observed, and factors which affect toxin lag periods are studied. The results show that the inclusion of B chains in conjugates increases ki values 3-10-fold, but only if the B chain is homologous with the A chain. In spite of the augmentation of antibody-toxin-conjugate ki values by homologous toxin B chain, these ki values are only 1/20 those observed with unmodified toxins on sensitive cells. A further difference noted between toxins and antibody-toxin conjugates is the presence of a dose-dependent lag when toxins, but not antibody-toxin conjugates, effect sensitive cell types. This lag period for ricin can be shortened by alkalinizing the cell medium. The kinetic data can be fit by assuming a processing step interposed between the binding of ricin to surface receptors and the interaction of the A chain with ribosomes which is first-order in toxin concentration and pH-dependent. The time constant of this event is reflected in the dose-dependent lag period. It is proposed that antibody-toxin conjugates do not participate in this processing event and therefore fail to achieve the high entry levels exhibited by unmodified toxins.     

Entering neurons: botulinum toxins and synaptic vesicle recycling

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release through their cleavage of components of the exocytosis machinery. These toxins are used to treat human diseases that are characterized by hyperfunction of cholinergic terminals. Recently, evidence has accumulated that gangliosides and synaptic vesicle proteins cooperate to mediate toxin binding to the presynaptic terminal. The differential distribution of synaptic vesicle protein receptors, gangliosides and toxin substrates in distinct neuronal populations opens up the possibility of using different serotypes of botulinum toxins for the treatment of central nervous system diseases caused by altered activity of selected neuronal populations.     

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.     

Bacterial glycosyltransferase toxins

Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification,cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.     

Structural characterization of the cell wall binding domains of Clostridium difficile toxins A and B; evidence that Ca2+ plays a role in toxin A cell surface association

Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.