The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm.     

环亚胺毒素gymnodimine的研究进展

Gymnodimine(GYM)是1994年从新西兰牡蛎中被鉴定出的藻毒素.其由凯伦藻（Karenia selliformis）产生,结构中含有一个位于螺环上的亚胺氮,属于环亚胺毒素.亚胺是GYM的毒理功能基团,具有很高的小鼠腹腔注射急性致死毒性,口服毒性很小,但详细的毒理作用机制尚不清楚.本文基于有限的研究资料,系统综述了GYM的结构、来源生物、毒性机理、携带生物、地理分布、降解代谢、剂量响应关系及风险评估等研究现状,并对今后藻毒素的重点研究方向进行了展望.     

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.     

Vibrio cholerae: cholera toxin

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.     

Attenuation of inhibitory processes in the central nervous system by tetanus toxin: an in vitro study on rat hippocampal slices

The influence of tetanus toxin on the efficiency of recurrent inhibition in the rat hippocampal slice was tested. The efficiency of the recurrent inhibition diminished in a dose-dependent manner following incubation of the slices with tetanus toxin. The effect was not observed in the slices preincubated for 3 hours with neuraminidase from Vibrio cholerae. This treatment reduces markedly the level of polysialogangliosides (receptor for tetanus toxin). It is concluded that tetanus toxin influences the efficiency of some inhibitory synapses in the central nervous system and that a certain level of polysialogangliosides is necessary for tetanus toxin to exert its action.     

From structure to solutions: the role of basic research in developing anthrax countermeasures: Microbiology Graduate Program Seminar: Anthrax toxin

Dr. John Collier traced the discoveries that elucidated the structure and function of the anthrax toxin in his talk "Anthrax Toxin," which was part of the Microbiology Graduate Program Seminar Series at Yale School of Medicine on February 23, 2012. Dr. Collier, Professor of Microbiology and Immunobiology at Harvard University, began by noting the advantages to studying anthrax pathogenesis in a biosafety level-1 lab. This designation does not merely facilitate his research, but also reflects a larger trend of basic research being leveraged to develop translational applications. Basic research on toxin structure has led to the development of a vaccine by Dr. Collier's group. Next-generation prophylactics also may stem from recent discoveries uncovering a role for cellular cofactors that mediate toxin function. Finally, basic research into the toxin substructure has facilitated efforts to change the receptor tropism to target dysregulated cells for therapeutic purposes. The urgency around biodefense agents makes the choice of research priorities a salient issue. As such, this author submits that basic research occupies a unique and lucrative niche driving clinical applications.     

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties.

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.     

Affinity of pertussis toxin produced by Bordetella pertussis for human haptoglobin: application to the in vitro assay of the toxin

Pertussis toxin formed a stable complex with human (Hp). Hemagglutinating activity of the toxin was inhibited in the presence of Hp, but leukocytosis activity was not.An enzyme linked immunosorbent assay for the toxin, Hp-ELIS, was developed on the basis of its specific affinity for Hp. A polystyrene microplate coated with Hp was incubated with samples containing the toxin. The bound was measured by sequential reaction with antipertussis toxin goat IgG, alkaline-labelled anti-goat IgG and p-nitrophenylphosphate.The Hp-ELISA method showed high specificity, high sensitivity and good correlation with leukocytes promoting activity in vivo. One ng of pertussis toxin could be detected within 3 h.     

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays.

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

Tetanus Toxin in Dissociated Spinal Cord Cultures: Long-Term Characterization of Form and Action

The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell-associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half-life of 5-6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1-week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.     

Identification of the characteristics that underlie botulinum toxin potency: implications for designing novel drugs

Botulinum toxin is a uniquely potent substance whose natural site of action is the peripheral cholinergic nerve ending. A substantial amount of information on the cellular, subcellular and molecular aspects of toxin action has been accumulated, and as a result a sound understanding of the basis for toxin potency has been developed. The principal characteristics of the toxin molecule that account for its potency are its ability: a) to be absorbed from the gut with minimal degradation; b) to bind to receptors that maximize the prospects of a pathophysiologic outcome; c) to act by a multiplicative (viz., enzymatic) mechanism; and d) to modify a substrate that is essential for neuronal function. Interestingly, the same properties that account for potency can also be exploited to utilize the toxin as a research tool and as a therapeutic agent. Several specific examples of ways to use the toxin advantageously are presented, including: a) development of oral medications and vaccines; b) analysis of subcellular mechanisms that govern transcytosis; c) identification of cell surface markers characteristic of cholinergic nerve endings; and d) analysis of specific aspects of exocytosis, such as spontaneous quantal release and synchronous quantal release. In all likelihood, further studies on the mechanism of botulinum toxin action will reveal yet further opportunities for utilizing it as a research tool or therapeutic agent.     

Mechanism of action of Clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells

Toxin B, an exotoxin produced by Clostridium difficile, induces the rounding-up and arborization of cultured mammalian cells, a typical effect which resembles that provoked by cytochalasins. In this study, the effect of toxin B was examined on astroglial cells grown in primary culture. A specific antiserum to toxin B was used to investigate its mechanisms of action. We found that the toxin exerts its effects on cell morphology after its incorporation into cells. The internalization of toxin B requires the presence of calcium ions in the extracellular medium. Replacement of NaCl with sucrose or with potassium glutamate prevents the internalization of the toxin. The direct introduction of calcium ions into cells by the calcium ionophore A23187 stimulates toxin-induced morphological changes. In contrast, toxin-induced morphological transformations were prevented in cells treated with tumor-promoting phorbol. esters or with dibutyryl-cAMP, although such treatment did not abolish the internalization of the toxin. As in the other cell types, the earliest effect of toxin B on astrocyte cytoskeleton is the disruption of actin filaments, without no visible alteration of intermediate filament nor microtubule networks. As astrocytes with toxin-induced stellate morphology survive toxin treatment, the progression of cell morphology and cytoskeleton organization were followed for several weeks. Twenty-six days after exposure to toxin B, stellate astrocytes have processes which were markedly longer and much more branched than those of cells freshly exposed to toxin. At that time, cells are still devoid of F-actin as assessed with rhodamine-conjugated phalloidin and only 70% contain vimentin while all astrocytes present in control cultures express vimentin. Some flat epithelioid astrocytes with prominent bundles of microfilaments reappear during the second week after toxin treatment. Our results show that Clostridium difficile toxin B is internalized into brain astrocytes in culture where it acts by modifying cytoskeletal elements. Its cytopathic effects are reversible. Although actin-related components of the cytoskeleton are the major target of toxin B, other cytoskeletal elements also seem to be affected.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.     

Effect of farm management practices in the Bt toxin production by Bt cotton: evidence from farm fields in China

Based on farm field plot level survey data and laboratory test, we examine the determinants of the expression of Bt toxin in China’s Bt cotton production. The results show that the expression of Bt toxin differs significantly among varieties. Even for the same variety the expression of Bt toxin also varies substantially among villages and among farmers in the same village. Econometric analyses show that after controlling for the effects of varieties and locations (or villages), farm management, particular applications of phosphate and potash fertilizers, and manure, has significant positive effects on Bt toxin expression in farmer’s fields. In contrast to previous studies which showed that nitrogen fertilizer has a positive impact on expression of Bt toxin, this study shows that nitrogen fertilizer has no significant impact on expression of Bt toxin in farmer’s fields. On the other hand, the expression of Bt toxin has a positive relationship with phosphate fertilizer, potash fertilizer and manure application.     

Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.     

Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.     

Purification and physicochemical properties of a unique Vero cell cytotoxin from Escherichia coli O157:H7

A unique Vero cell cytotoxin has been purified to homogeneity from a strain of Escherichia coli O157:H7, using ultrafiltration with Pellicon membrane cassettes and chromatography with QAE Sephadex A-50. SDS-PAGE showed the molecular weight of the toxin to be 64,000 and the absence of subunits. Based on analytical isoelectric focusing, the toxin had pI of 5.2. This Vero cell toxin was lethal to mice and showed pathological abnormalities of the mouse colonic mucosa when administered intraperitoneally. Vero cell cytotoxicity of this toxin was not neutralizable with rabbit antiserum to Shiga toxin. Based on physicochemical and immunological properties, this toxin is different from the Shiga-like toxin previously found in this organism.