A Potential Epidemic Factor from the Bacteria, <Emphasis Type="Italic">Vibrio cholerae WO7</Emphasis>

Certain species of Vibrio cholerae have evolved mechanisms to become pathogenic to humans, with the potential to cause a severe life-threatening diarrheal disease, cholera. Cholera can emerge as explosive outbreaks in the human population. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. The present study has been carried out on a novel toxin purified from V. cholerae W07, an epidemic cholera strain devoid of cholera toxin gene (ctx). A modified method of purification improved purification fold as well as yield of this toxin. Heating was found to be the essential and sufficient condition for dissociation of the two subunits (58 kDa and 40 kDa) of this toxin (pI 5.2). The 40-kDa subunit of the purified toxin was identified as the carbohydrate binding subunit. This toxin was found to induce apoptosis in HEp-2 cells. Thus, the WO7 toxin seems to have potential importance in the pathogenesis of disease associated with Vibrio cholerae WO7.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Selection of a <Emphasis Type="Italic">Clostridium perfringens</Emphasis> type D epsilon toxin producer via dot-blot test

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.     

Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae

A new toxin of Enterobacter cloacae able to lyse erythrocytes and leukocytes was found. Purification of the toxin was performed by salt precipitation, gel filtration, ion exchange and HPLC in C₈ column. SDS-PAGE electrophoresis showed more than one bank corresponding to the leukotoxin able to form polymers and aggregate like some pore-forming cytotoxins (RTX). In culture supernatant the toxin showed 1 HU/ml (hemolytic unit) and 1.5 LU/ml (leukotoxic unit); after purification it reached 15 HU/ml and 20 LU/ml. The ratio between HU and percentage red cells affected the lytic capacity. E. cloacae toxin stimulated the oxidative metabolism of neutrophils, but over 50 μg toxin/ml the stimulus ceased as it was shown by NBT assay due to cell death. Chemiluminescence evidenced an increase in superoxide anion generation, but an excess of toxin interfered with this stimulus, as was previously observed in HlyA Escherichia coli toxin. Cross-reaction was found by immunoblotting with this HlyA. E. cloacae toxin presented higher amounts of proline, valine, aspartic and glutamic acids than HlyA. E. cloacae toxin was similar to HlyA in the prescence of a glycine-rich DNA sequence and in the observed effect of calcium on toxin activity. E. cloacae toxin did not cross-react by immunoblotting with hemolysin HmpA of Proteus. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Sandwich Enzyme-Linked Immunosorbent Assay for the Bacillus sphaericus Binary Toxin

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5α harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362. Received: 2 July 1997 / Accepted: 12 August 1997     

High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants

Ustilago maydis killer toxins are small polypeptides (7–14 kDa) whichkill susceptible cells of closely related fungal species. The KP4 toxin is a single polypeptide subunit with a molecular weight of 11.1 kDa. In this work, a transgenic tobacco plant was constructed which secretes the KP4 toxin at a high level. The KP4 toxin expressed in this transgenic plant was of the same size and specificity as the authentic Ustilago KP4 toxin. The expression level was at least 500 times higher than that of the KP6 toxin expressed in plants. Transgenic crop plants producing the KP4 toxin could be rendered resistant to KP4-susceptible fungal pathogens.     

Differential expression of defense‐related proteins of rice and sheath blight symptoms in response to active and inactive Rhizoctonia solani toxin

The effects of the phyotoxin from the fungal pathogen Rhizoctonia solani, causing sheath blight on the expression of defense‐related proteins of rice were investigated. The toxin inactivated by chemical treatment and by the toxin‐inactivating enzyme α‐glucosidase produced by Trichoderma viride was used in the study along with the active toxin. Toxin inactivated by T. viride α‐glucosidase and sodium periodate caused significantly less damage and electrolyte leakage to test plants. The active toxin and the pathogen induced chitinase and ß‐1,3‐glucanase synthesis in rice plants, while the inactivated toxin did not have any effect on the expression of these pathogenesis‐related proteins. The toxin was found to suppress the peroxidase activity 72 h after inoculation and the inactivated toxin restored the activity as that of untreated plants. There was no remarkable change in phenylalanine ammonia lyase activity in rice sheath treated with both the forms of the toxin.     

Characterization of a toxin produced by a rhizobacterialPseudomonas sp. that inhibits wheat growth

A toxin produced by a deleterious rhizobacterial pseudomonad that inhibits both winter wheat (Triticum aestivum L.) root andEscherichia coli growth was characterized. The toxin was rapidly deactivated at pH 2 and 12 and by autoclaving (121°C, 15 minutes). Less toxin was destroyed as the temperature and time of exposure decreased, and at 40°C it was stable for at least 24 hours. The toxin was extremely polar and could not be extracted from culture filtrates with organic solvents. The compound eluted after the void volume from a Sephadex G-10 column indicating a molecular weight of less than 700. The toxin adsorbed to Dowex 50W strong cation exchange resin and eluted with 2M NH₄OH. Numerous thin layer chromatography solvent systems were unsuccessful at purifying the toxin. The partially purified toxin inhibited several different microorganisms while the producing strains were resistant. The toxin appears unique to toxins produced by recognized plant pathogenic bacteria.Contribution from the Agric. Res. Serv., U.S. Dept. of Agriculture in cooperation with the College of Agric. and Home Econ., Res. Ctr., Washington State University, Pullman, WA 99164, USA     

Recombinant Enterobacter amnigenus Highly Toxic to Anopheles dirus Mosquito Larvae

The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.     

Accumulation of a peptide toxin from the cyanobacterium Oscillatoria agardhii in the freshwater mussel Anadonta cygnea

Swan mussels (Anodonta cygnea) were exposed to a toxic strain of the cyanobacterium Oscillatoria agardhii. Mussels accumulated large amounts of the peptide Oscillatoria toxin which was present in low concentrations within the cyanobacterial cells in the test aquaria (40–60 µg Oscillatoria toxin/1). The toxin concentration in the mussels increased during the experiment and after 15 days of exposure the concentration was 70 ± 2 µg/g freeze dried tissue (mean ± range of values). The highest concentration of the toxin (130 µg/g of freeze dried tissue) was found in the hepatopancreatic tissue. The toxin did not seem to be metabolized in the mussels and they were not killed by the high toxin concentrations within them. After two months in clean water still detectable amounts of toxin were present in the mussels.     

致命鹅膏菌毒素对DMBA/巴豆油诱导小鼠皮肤肿瘤的治疗作用

研究了致命鹅膏中的毒素在安全剂量下抑制小鼠皮肤肿瘤的效果,并对此毒素的药用安全性进行初步评价。采用经典的DMBA/巴豆油诱导小鼠皮肤乳头状瘤形成的二阶段致癌模型,观察和评价致命鹅膏毒素在不同剂量下抑制肿瘤的效果,并且通过测定血清中氨基己糖的含量来判断对肝损伤的情况。结果表明,致命鹅膏毒素在某一低剂量下对DMBA/巴豆油诱导小鼠皮肤乳头状瘤有较好的治愈效果,且对小鼠肝脏无损伤,证明了致命鹅膏毒素可以较好地治疗皮肤癌。     

Bt toxin is not taken up from soil or hydroponic culture by corn,carrot, radish,or turnip

The culture of transgenic Bt corn (Zea mays L.) has resulted in concern about the uptake of the Cry1Ab protein toxin by crops subsequently grown in soils in which Bt corn has been grown. The toxin released to soil in root exudates of Bt corn, from the degradation of the biomass of Bt corn, or as purified toxin, was not taken up from soil, where the toxin is bound on surface-active particles (e.g. clays and humic substances), or from hydroponic culture, where the toxin is not bound on particles, by non-Bt corn, carrot (Daucus carota L.), radish (Raphanus sativus L.), and turnip (Brassica rapa L.). The persistence of the toxin in soil for 90 days after its addition in purified form or for 120–180 days after its release in exudates or from biomass, the longest times evaluated, confirmed that the toxin was bound on surface-active particles in soil, which protected the toxin from biodegradation. The greater toxicity of the toxin in soil amended with 9% montmorillonite or kaolinite than in soil amended with 3% of these clay minerals indicated that the binding and persistence of the toxin increased as the clay concentration was increased.     

Processing of the maize Bt toxin in the gut of Mythimna unipuncta caterpillars

Mythimna unipuncta Haworth (Lepidoptera: Noctuidae) is a well‐known moth species whose larvae can cause devastating damage to some Poaceae crops, including maize (Zea mays L.). The low susceptibility to the Bacillus thuringiensis Berliner (Bt) toxin observed in L6 larvae of this species has been the object of several studies. This study aimed to clarify whether the toxin eliminated from the content of the peritrophic membrane is degraded or excreted and whether the effects of the Bt toxin depend on the doses ingested. To this end, L6 larvae were fed on diets with different amounts of lyophilized Bt or non‐Bt maize leaves. The effect of the Bt concentrations on larval development was measured and the fate of the toxin in the larval tissues was tracked. Results indicated that the larvae of M. unipuncta fed on the various diets showed few differences in weight gain, duration of development, or pupal weight between sublethal Bt concentrations. The larvae rapidly excreted a large part of the toxin ingested, whereas inside the peritrophic membrane the toxin was eliminated, degraded, or sequestered at a rate that increased with the dose and the duration of feeding. As a consequence, little toxin reached the midgut epithelium and therefore the binding sites of the toxin. Moreover, larvae fed on the Bt toxin recovered quickly when they were transferred to a non‐Bt diet.     

Rhizobitoxine: A phytotoxin of unknown function which is commonly produced by bradyrhizobia

Summary Fifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America. Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine. Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin. Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj₁ rj₁) soybeans.     

Effect of Clostridium perfringens Alpha Toxin on Contraction of Isolated Guinea-Pig Diaphragm

The effect of Clostridium perfringens alpha toxin on contraction induced by-electric stimulation of isolated guinea-pig diaphragm was investigated. The toxin inhibited electrically stimulated contraction of the tissue in a dose- and incubation time-dependent manner. Tetrodotoxin resulted in no effect of the action of the toxin. Nifedipine dose-dependently delayed the action of the toxin, but verapamil and diltiazem did not. On the other hand, treatment of the toxin with N-acetylimidazole caused significant reduction of the inhibitory activity of the toxin on contraction, but did not cause significant loss of phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine. The data showed that the toxin impairs contraction of isolated guinea-pig diaphragm.     

Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin

Toxin A, one of several virulence factors secreted by the gram-negative bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precursor with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside within the region defined by the toxin A LP and the first 30 amino acids (aa) of mature toxin A. In the present study, we have used exonuclease Ba131 deletion analysis to examine the specific role of the first 30 as in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue region fused to a toxin A carboxy-terminal region, were identified. In addition, a gene fusion encoding a hybrid protein consisting of the LP of P. aeruginosa elastase and the final 305 residues of toxin A, was generated. The cellular location of the toxA subclone products in P. aeruginosa was determined by immunoblotting analysis. Toxin A CRMs (cross-reacting material) encoded by different subclones were detected in different fractions of P. aeruginosa including the periplasm and the supernatant. Results from these studies suggest that (1) mature toxin A contains two separate secretion signals one within the N-terminal region and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.     

Zearalenone production in barley

A plot of barley cv. Golden Promise was sprayed with an isolate of Fusarium culmorum known to produce zearalenone, an oestrogenic toxin. The grain was harvested and stored under conditions that were known to induce toxin production in sterilized grain inoculated with the same strain. Toxin was not found in the harvested grain but appeared 20 wk after storage. A plot of unsprayed barley was harvested at the same time as the sprayed plot. F. culmorum was isolated from the grain at the time of harvesting but there was no toxin detectable. This appeared at the same time as toxin in the sprayed plot. Higher concentrations of zearalenone were produced in the control suggesting that the F. culmorum isolated was either a different strain from that used to inoculate the sprayed plot or that the microbial flora of the grain affected toxin production.     

A shared epitope found in the major paralysis inducing tick species of Africa

Cross-reactivity between all the paralysis inducing tick species of veterinary relevance in Africa was demonstrated, by using a monoclonal antibody directed against the paralysis inducing toxin of Rhipicephalus evertsi evertsi. Western blot results, together with amino acid composition studies indicated that this monoclonal antibody recognizes protein bands of similar molecular mass and amino acid composition in R. evertsi evertsi and Ixodes rubicundus. This suggests that the Karoo paralysis toxin of I. rubicundus is possibly also a trimer with a high degree of homology to the spring lamb paralysis toxin of R. evertsi evertsi. The conclusive identity of these protein bands of 1. rubicundus could not be shown. Bio-assay studies performed on 1-day-old chickens suggested that the anti-spring lamb paralysis toxin monoclonal antibody also recognizes the paralysis toxin present in Argas (Persicargas) walkerae, by rendering some degree of protection against the effect of this toxin.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.