Identification of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains. These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.     

Identification of Renibacterium salmoninarum surface proteins by radioiodination

Abstract Treatment of Clostridium perfringens alpha toxin with aminopeptidase resulted in no effect on various activities of the toxin. Aminopeptidase did not hydrolyze the native toxin or the toxin treated with urea in the presence of EDTA. Treatment with carboxypeptidase for 30 min resulted in a 75% decrease in these activities. Incubation of the native toxin with carboxypeptidase for 30 min released approximately 15 amino acids from the C-terminus of the toxin. The biological activities of a mutant toxin lacking 20 C-terminal residues of the toxin (AT_1–350) showed about 59–87% of the activity of native toxin. The mutant toxin showed partial antigenic identity with the native toxin. These data suggest that the C-terminal domain contributes to maintaining the active form of the toxin.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells

A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 degrees C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 degrees C, and the cells were then shifted to 37 degrees C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 125I-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 125I-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of 125I-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7.     

Proteolytic Cleavage of Tetanus Toxin Increases Activity

Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single-chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two-chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two-chain toxin is demonstrated to have greater activity than the single-chain toxin preparations. Indeed the activity of the single-chain toxin preparations can be explained by the small amounts of residual two-chain toxin present in these extracts. Using a protease from a toxin-minus strain of Clostridium tetani to convert a single-chain toxin preparation to two-chain toxin increases toxin activity. In vivo the single-chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single-chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure-function relationship similar to that of other bacterial protein toxins.     

Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates

We describe a comprehensive detection system for 18 kinds of classical and newly described staphylococcal superantigenic toxin genes using four sets of multiplex PCR. Superantigenic toxin genotyping of Staphylococcus aureus for 69 food poisoning isolates and 97 healthy human nasal swab isolates revealed 32 superantigenic toxin genotypes and showed that many S. aureus isolates harbored multiple toxin genes. Analysis of the relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements suggests its possible role in determining superantigenic toxin genotypes in S. aureus as combinations of toxin gene-encoding mobile genetic elements.     

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

Metabolism of trichothecene mycotoxins. I. Microsomal deacetylation of T-2 toxin in animal tissues

In an attempt to elucidate the active form of T-2 toxin, one of trichothecene mycotoxins in vivo, the metabolism in animal tissues was studied in vitro by using gas liquid chromatography. T-2 toxin was selectively hydrolysed by the microsomal esterase at C-4, giving rise to HT-2 toxin as the only metabolite. This esterase activity was found mainly in the microsomes of liver, kidney, and spleen of laboratory animals. Since the enzymatic hydrolysis of T-2 toxin was inhibited by eserine, and diisopropylfluorophosphate, it is concluded that non-specific carboxyesterase [EC 3.1.1.1] of microsomal origin participates in this type of selective hydrolysis of T-2 toxin. The microsomal fraction from rabbit liver was proved to be a convinient material for the preparation of HT-2 toxin from T-2 toxin. From the evidence that the toxicity of HT-2 toxin is comparable to that of T-2 toxin and that the microsomal fraction of whole liver possesses the ability to biotransform the total lethal dose of T-2 toxin into HT-2 within a few minutes, T-2 toxin administered to animals is presumed to exhibit its toxicity partly as HT-2 toxin.     

Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity

Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A. DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A. RNAase had no effect on either toxin. The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.     

Assay and Relationship of HT-2 Toxin and T-2 Toxin Formation in Liquid Culture

Both T-2 toxin and HT-2 toxin can be conveniently quantitated in crude extracts by using a combination of thin-layer chromatography and fluorodensitometry. This technique was used to follow the production of these toxins by liquid cultures of Fusarium poae (NRRL 3287). T-2 toxin was produced prior to HT-2 toxin and hexadeuterio-T-2 toxin was converted by the culture to trideuterio-HT-2 toxin.     

Structural requirements for furin-induced cleavage and activation of Shiga toxin

Shiga toxin has a protease-sensitive site in the disulfide loop region of the A-chain. Cleavage of this site by furin is essential for rapid intoxication of cells by Shiga toxin. We have here investigated whether in addition to the Arg-X-X-Arg sequence, there are other structural requirements in the disulfide loop region for furin cleavage. A toxin mutant (Shiga-2D toxin) still containing the consensus motif for cleavage by furin, but lacking ten amino acids in the disulfide loop, was generated. Trypsin was able to cleave Shiga-2D toxin in vitro, demonstrating that the protease-sensitive region is intact. However, Shiga-2D toxin was not efficiently cleaved by furin either in vitro or in vivo. Furthermore, unless it was precleaved with trypsin, Shiga-2D toxin was much less toxic than wild type Shiga toxin in LoVo cells expressing functional furin. In contrast, LoVo/neo cells lacking functional furin were unable to activate both wild type Shiga toxin and Shiga-2D toxin. In conclusion, an extended loop structure is required for furin-induced cleavage of Shiga toxin.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

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.     

Pharmacological effect of beta toxin of Clostridium perfringens type C on rats

The biological effect of purified beta toxin of Clostridium perfringens type C in vivo was investigated. After intravenous injection of the purified beta toxin into rats, a rise in blood pressure and a simultaneous fall in heart rate were observed. After the blood pressure reached a maximum, the heart rate recovered gradually, and electrocardiographic and respiratory changes began. The rise in blood pressure induced by beta toxin tended to be proportional to the amount of toxin. The latent period between the injection of toxin and the onset of the increase, and also the time between the injection and the maximum pressure induced by the toxin decreased with increasing concentration of the toxin. A good correlation was found between the factor producing the rise in blood pressure and beta toxin. Alpha adrenergic and ganglionic blocking agents reduced blood pressure levels elevated by beta toxin. The data suggest that the toxin causes a release of catecholamines, and that the increase in blood pressure was induced by released catecholamines.     

The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxin

Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin-receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.     

Entry of diphtheria toxin-protein A chimeras into cells

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.     

Characterization of tetanus toxins and toxin components by amino terminal analyses

Extract tetanus toxin, filtrate tetanus toxin, and the heavy and light chains of filtrate toxin were analyzed for their amino termini with 4-N,N-dimethylaminoazobenzene-4′isothiocyanate and phenylisothiocyanate. Extract toxin (intracellular toxin) is a single-chain polypeptide with proline as the amino terminus. Filtrate toxin (extracellular toxin) is a mixture of species produced by endogenous proteases, and showed three major amino terminal residues, proline, asparagine, and serine. Cleavage points in the filtrate toxin molecule appear to be on either side of a disulfide bond. Reductive and nonreductive preparative electrophoresis of filtrate toxin produce different species of light and heavy chains. The light chains have a single amino terminus of proline, indicating that the light chain is the amino terminal portion of the toxin molecule. The heavy chains showed no proline but rather asparagine and serine as the major amino termini. Small amounts of other amino terminal residues were present, indicating microheterogenity at the cleavage sites in the toxin. The results permit the construction of a model of tetanus toxin which is consistent with the fragments obtained from either reductive or nonreductive preparative electrophoresis of filtrate toxin.     

Production and characteristics of monoclonal antibodies to the diphtheria toxin

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.