A yeast bioassay for T-2 toxin

A disk diffusion type bioassay was developed for T-2 toxin using the yeast Kluyveromyces fragilis. The lower limit of detection for this in 0.2 μg of T-2 toxin. The growth of this yeast was sensitive to other trichothecenes such as verrucarin A (0.01 μg). Aflatoxin B₁ (50 μg) and zearalanone (20 μg) did not inhibit the growth of this yeast.     

Antibacterial Activity of Sirodesmin PL Phytotoxin: Application to the Selection of Phytotoxin-Deficient Mutants

Sirodesmin PL, a phytotoxin and mycotoxin produced by Leptosphaeria maculans, the causal agent of stem-canker disease of crucifers, exhibited antibacterial activity against gram-positive bacteria and particularly Bacillus subtilis. The importance of the disulfide bridge of the molecule in antibacterial activity was demonstrated. A simple and reliable bioassay based on the antibacterial activity of the toxin was performed for screening sirodesmin PL-deficient mutants when grown on solid culture medium. A mutant was selected and found to produce 3,700-fold less toxin than did the wild-type strain. A sensitive procedure for quantification of the toxin by high-pressure liquid chromatography was developed. Levels of product as low as 100 ng could be detected by this procedure.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn,Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 μg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).     

The detection of a deletion in the type B neurotoxin gene of Clostridium botulinum A(B) strains by a two-step PCR

Differences between the type B neurotoxin gene sequence of Clostridium botulinum type A(B) and Cl. botulinum type B, including a six nucleotide deletion, were recently proposed as a cause of the lack of expression of this gene in the type A toxigenic strains. A polymerase chain reaction (PCR) based on two sets of primers was designed to investigate the absence of the 6-nucleotide sequence in the apparently unexpressed type B toxin gene of 42 strains of Cl. botulinum type A(B). Thirty-five strains were shown to exhibit a deletion in their type B toxin gene; two strains did not have the deletion and actually produced small amounts of type B toxin when tested by the mouse bioassay. This two-step PCR might be useful for the rapid determination of the presence of the six nucleotide deletion and consequently, whether the type B toxin is likely to be produced.     

Eyespot Disease of Sugarcane : INDUCTION OF HOST-SPECIFIC TOXIN AND ITS INTERACTION WITH LEAF CELLS

Helminthosporium sacchari produces a toxin which is responsible for the symptoms of eyespot disease in Saccharum officinarum. A rapid and highly repeatable bioassay based on increase in conductivity of tissue leachates showed that the interaction of toxin with sugarcane obeys Michaelis-Menten hyperbolic saturation kinetics. There was no evidence for positive or negative cooperation interaction. Resistant and susceptible cultivars of sugar cane had distinctive conductivity characteristics. Co-cultures of H. sacchari and suspension cultures of sugarcane gave up to a 4,000-fold increase in toxin production.     

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV by tyrosine nitration

Reaction of Cerebratulus lacteus toxin B-IV with tetranitromethane in the presence of low concentrations of urea results in essentially complete loss of toxicity as measured by a sensitive quantal bioassay. Amino acid analysis and speetrophotometric studies both indicate the primary effect of reaction to be nitration of a single tyrosine residue per molecule of toxin. The nitrated residue has been identified as tyrosine-9 by automated Edman degradation of the modified protein. Since the secondary structure of toxin B-IV is not detectably altered by nitration, it is concluded that tyrosine-9 is directly involved in the interaction of this polypeptide with its axonal receptor, proposed to be involved in the inactivation of voltage-sensitive Na⁺ channels in crustacean nerves.     

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.     

Isolation, Purification, and Characterization of a Killer Protein from Schwanniomyces occidentalis

The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40°C. The killer protein was chromosomally encoded. Mannan, but not β-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.     

Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

     

Variety of PSP toxin profiles in various culture strains of Alexandrium tamarense and change of toxin profile in natural A. tamarense population

Paralytic shellfish poisoning (PSP) toxin profiles were compared between clonal and axenic culture strains of Alexandrium tamarense prepared from cysts. The cysts were collected from two stations in northern Japan. The major toxin components of A. tamarense were C2 and GTX4, however, the proportions of C2 and GTX4 varied largely 0.7-78.8 mol% and 79.4-8.5 mol%, respectively. Some culture strains contained significantly higher proportion of neoSTX than other strains. These results indicate that strains with various toxin profiles exist in the same region, and suggest that the comparison of the toxin profiles between strains at different localities is considerably difficult. A drastic change of the toxin profile was observed in natural planktonic populations containing A. tamarense. This may be explained by the presence of a lot of planktonic populations with various toxin profiles growing around the sea area.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Assessment of changes in the epidemiology of Clostridium difficile isolated from diarrheal patients in Hungary

150 Clostridium difficile strains isolated from diarrheal feces were collected from three parts of Hungary and the presence of genes responsible for toxin A and B, and binary toxin production were examined. MIC distribution against clindamycin, erythromycin, metronidazole, moxifloxacin and rifampin of 80 toxigenic strains selected from the above-mentioned strains and 20 large clostridial toxins (LCTs)-positive strains chosen from our earlier strain collection were determined. 80% of the examined 150 strains were positive for both tcdA and tcdB, and no toxin A-negative, toxin B-positive isolates were found during the study period. 5.3% of toxigenic strains proved to be positive for binary toxin too. Among binary toxin-positive strains, one strain showed the same pattern characteristic of PCR ribotype 027. Comparison of recent findings and our earlier results, the prevalence of toxin-producing and binary toxin-positive strains among C. difficile isolated from diarrheal specimens increased. No metronidazole resistant isolate was detected among strains isolated in 2002–2003 and 2006–2007. The rates of resistance to erythromycin, clindamycin, moxifloxacin and rifampin among strains isolated between 2006 and 2007 were 25%, 27.5%, 25% and 6.3%, respectively. Erythromycin resistance was frequently associated with clindamycin and moxifloxacin resistance, however this resistant phenotype was not found among strains isolated in 2002–2003.     

Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A

Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of C. botulinum type A neurotoxin.     

Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.     

Genomic Relatedness of Clostridium difficile strains from different toxinotypes and serogroups

Clostridium difficile strains can be divided into sixteen toxinotypes (0 and I to XV) according to changes in their toxin genes. To determine the genomic similarity between toxinotypes, two molecular typing techniques were used, AP-PCR and PFGE. Strains were selected from five serogroups (A1, A15, E, F, X) and represented non-toxinogenic isolates, strains with toxin genes identical to the reference C. difficile strain, VPI 10463 (toxinotype 0), and strains with variant toxin genes from toxinotypes III, IV, V, VI, VII, VIII, IX, and XI. The strains studied formed three main clusters, which correlated well with serogroups: in the first were strains from serogroup A15 and E; in the second, serogroup A1 strains; and in the third, strains from serogroups F and X. Within these three clusters strains of a single toxinotype were grouped together. Toxinotypes III, IV and VIII were more similar to strains with ordinary toxin genes or non-toxinogenic isolates within the same serogroup than to other toxinotypes. Toxinotypes V, VI, VII, and XI, which exhibit similar changes in their toxin genes, seem to be more closely related one to another than to other toxinotypes. It can be concluded that variant Clostridium difficile strains do not have a common ancestor and that groups of different toxinotypes arose independently from strains with ordinary toxin genes.