Our experiences with isolation of heat-labile enterotoxin from Escherichia coli

From the Escherichia coli strain isolated from a patient suffering from diarrhoea a homogenate and concentrated culture filtrate were prepared. From these materials the heat-labile enterotoxin was isolated after its elution with 0.2 M D-galactose from Sepharose 6B column. The obtained enterotoxin was positive in the rabbit ileal loop test up to a concentration of 1 microgram protein/ml. In the immunodiffusion test it reacted in a concentration up to 5 micrograms protein/ml with anticholeragen and in a concentration up to 30 micrograms protein/ml with its specific antiserum. This antiserum was prepared by intramuscular immunization of rabbits by enterotoxin with complete Freund's adjuvant.     

Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs

Summary The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM₁ considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin Bor staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.     

Pyrogenic Responses to Staphylococcal Enterotoxins A and B in Cats

Pyrogenic responses, ranging up to 4.8 F, were induced in cats by oral administration of highly purified staphylococcal enterotoxin B in doses from 10 to 100 mug/kg. Fever was a more sensitive indicator of intoxication than was emesis. Highly purified preparations of enterotoxin A, whether administered intravenously (0.01 to 1.0 mug/kg), orally (10 to 25 mug/kg), or into the cerebral ventricles (0.005 to 0.020 mug in 0.20 ml), were also pyrogenic in cats. Tolerance to the pyrogenic activity was produced by repeated intravenous injection of a given dose of enterotoxin A but not by repeated intracerebroventricular injection. Enterotoxin A was more potent than enterotoxin B after intravenous injection in causing both fever and emesis. Cross-tolerance could not be demonstrated between enterotoxin A and enterotoxin B or Salmonella typhosa endotoxin. This lack of cross-tolerance plus the inability of large oral doses (100 to 4,700 mug/kg) of endotoxin to cause fever or emesis indicate that the reported responses were attributable to the specific toxins administered and not to contamination by other pyrogens.     

Structural similarity and functional activity of molecular fragments of staphylococcal enterotoxin B and thymopoietin II

Structural similarity between Staphylococcus enterotoxin B and a thymus hormone, thymopoietin, has been revealed concerning thymopentin, one of active centres of thymopoietin. Fragments of enterotoxin B homologous to thymopoietin and their analogues were synthesized their immunological activity studied. Acetyl derivative of the fragment 225-229 of enterotoxin B increased infectional resistance and stimulated delayed type of hypersensitivity reaction.     

霍乱毒素佐剂的研究进展

霍乱毒素（CT）是霍乱弧菌分泌的一种不耐热肠毒素,具有很强的免疫原性和佐剂活性,是当今研究得最多且最深入的粘膜免疫佐剂之一,CT本身有很强的毒副作用,而一定的毒性又似乎是发挥佐剂作用所必须的。通过各种改造,使之具备优良的佐剂活性而没有显的毒副作用是当前研究的主要目标。本从CT作为佐剂的作用机理出发,概述当前对其进行改造的几种研究方向。     

Resistance of Free-Living Nematodes to Staphylococcal Enterotoxin

The usefulness of free-living nematodes for assaying staphylococcal enterotoxin was evaluated with a 98% pure enterotoxin B on five different nematodes. Included in the evaluation was an enterotoxin B in a crude culture filtrate. The filtrate of a culture of nonenterotoxigenic strains of Staphylococcus aureus, the uninoculated respective broth media, and distilled water were used as controls. The purified enterotoxin was found to exert no toxic effects at dosages ranging from 10 to 1,000 μg/ml for as long as 24 hr. Utilization of the toxin-protein by these nematodes was evidenced by their propagation after exposure times longer than 24 hr. The crude filtrate, containing 28 μg of enterotoxin per ml, was detrimental to nematodes to the same degree as the nontoxic filtrate and the uninoculated broths, in that they all caused irritation to external genitalia, motility changes, and death after comparable exposure times. This is in agreement with earlier observations that standard bacteriological fluid media, or broths containing over 1% protein hydrolysate or 1 to 2% salts, exert toxic effects on free-living nematodes.     

Failure to Detect Cell-Associated Enterotoxin B in Staphylococcus aureus by Immunofluorescence

Enterotoxin B-producing and -nonproducing Staphylococcus aureus strains showed cell fluorescence when tested with fluoresceinisothiocyanate-labeled rabbit anti-enterotoxin B globulin, probably as a result of a protein A-immunoglobulin G (Ig G) interaction. No cell-bound enterotoxin B could be detected by immunofluorescence using F(ab(1))(2)-fragments of anti-enterotoxin B globulin. However, soluble enterotoxin B could be estimated by immunofluorescence. Approximately 1,000-fold more enterotoxin B was detected by immunodiffusion as an extracellular product in the media than could be detected in the cell fraction. The results show that intact Ig G is not suitable for the detection of antigens other than protein A on the cell surface of S. aureus in conventional immunofluorescence. For such purposes, the use of F(ab(1))(2)-fragments of Ig G is recommended.     

The complete amino acid sequence of staphylococcal enterotoxin C1

This report presents the complete amino acid sequence of staphylococcal enterotoxin C1. It has a total of 239 amino acids and a calculated Mr = 27,500. Reaction of the native toxin with trypsin produced five peptides, designated A through E. Their structures were determined after further fragmentation with cyanogen bromide, trypsin, and chymotrypsin. Overlap peptides were prepared by cleavage at the two half-cystine residues, and by the reaction of enterotoxin C1 with hydroxylamine. The latter procedure was employed when it was found that one of the three asparaginyl-glycine loci in the toxin is refractory to direct sequencing. The sequence is compared to staphylococcal enterotoxin B. Extensive homology is found, particularly in the carboxyl-terminal region. However, the segment spanned by the disulfide bond in enterotoxin C1 is three residues shorter than the corresponding segment in the B variant.     

Analysis of receptor-binding site in Escherichia coli enterotoxin

Heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and cholera enterotoxin are both composed of A and B subunits. The A subunit is an enzymatically active ADP-ribosylating subunit, while the B subunit, consisting of 103 amino acids, binds the toxin to a receptor, GM1-ganglioside, on the cell surface. A mutant isolated after treatment of E. coli producing heat-labile enterotoxin with N-methyl-N'-nitro-N-nitrosoguanidine produces a B subunit that is unable to bind to ganglioside. This subunit was purified and its primary amino acid sequence was determined. It differed from the native B subunit in only one amino acid at position 33; namely it had aspartate instead of glycine at position 33 from the N terminus. Thus glycine at position 33 from the N terminus of the B subunit is important for binding the B subunit to the ganglioside receptor.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

Production and characterization of monoclonal antibodies against the hemolysin BL enterotoxin complex produced by Bacillus cereus.

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L(2) component, and antibody 1C2 was specific for the L(1) protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Role of B7 costimulatory molecules in the adjuvant activity of the heat-labile enterotoxin of Escherichia coli

Much interest has been directed at understanding the adjuvant properties of the heat-labile enterotoxin of Escherichia coli (LT). In this study, we have assessed how LT compared with the nonenzymatic mutant LT (E112K) affect the level of B7-1 and B7-2 expression on APCs, and we determined how these costimulatory molecules influence their adjuvant properties. Analysis of B7-1 and B7-2 expression on B cells revealed that LT enhanced B7-2 but not B7-1, while LT (E112K) had no effect on the expression of either costimulatory molecule. Treatment of macrophage or dendritic cells with LT resulted in a predominant enhancement of B7-2, while LT (E112K) induced mainly B7-1 expression. Analysis of LT- and LT (E112K)-treated B cells, macrophage, and dendritic cells also revealed significant differences in their ability to enhance anti-CD3-stimulated CD4(+) T cell proliferative responses via B7-1 and B7-2. Furthermore, the ability of LT to enhance both Ab and CD4(+) T cell responses to a coadministered Ag was severely abrogated in B7-2- but not B7-1-deficient mice. In contrast, the in vivo adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4(+) T cell responses, while B7-1 appeared to be the predominant B7 molecule involved in the ability of LT (E112K) to augment Ab responses to a coadministered Ag. These findings demonstrate distinct differences in the ability of LT and LT (E112K) to enhance B7-1 and B7-2 on APC, as well as a dependence upon these costimulatory molecules for their adjuvant properties.     

大肠埃希菌热不稳定肠毒素B亚单位在婴儿双歧杆菌中的表达

目的将大肠埃希菌热不稳定肠毒素B亚单位（LTB）克隆到表达载体pBV220,使LTB基因在大肠埃希菌和双歧杆菌中稳定表达。方法将LTB基因克隆到表达载体pBV220中,再分别转化大肠埃希菌DH5a和婴儿双歧杆菌,使之表达,表达产物用SDS—PAGE鉴定。并通过家兔肠袢实验验证表达蛋白的安全性。结果LTB基因在大肠埃希菌和双歧杆菌中均可稳定表达,毒性实验证明LTB保留轻微的毒性。结论稳定表达LTB的双歧杆菌为今后的口服疫苗佐剂奠定了基础。     

Extrachromosomal Control of Methicillin Resistance and Toxin Production in Staphylococcus aureus

All of 41 naturally occurring coagulase-positive methicillin-resistant strains of Staphylococcus aureus isolated in various laboratories were resistant to several antibiotics and were lipase-negative. Most strains produced hemolysins, and 38 strains produced enterotoxin B. Acriflavine treatment of four strains resulted in elimination of resistance to methicillin and mercury; in one strain, resistance to cadmium was also lost. Production of enterotoxin B and beta-hemolysin was eliminated in all four strains and penicillinase production was eliminated in one strain. In transduction experiments, methicillin resistance and enterotoxin B production were transferred together at a frequency of 0.2 x 10(-8) to 1.1 x 10(-8) by use of ultraviolet-induced phage lysates from naturally lysogenic methicillin-resistant strains. Cotransductions of resistance to mercury and cadmium, as well as production of penicillinase and beta-hemolysin, were obtained to some extent. The extrachromosomal character of these determinants and their possible genetic association are discussed.     

Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.     

苏云金杆菌肠毒素基因的检测和全长基因的扩增

以煮沸冻融法制备PCR扩增模板,应用肠毒素（enterotoxin)基因特异引物EntA和EntB对15个苏云金杆菌（Bacillus thuringiensis,Bt)血清变种进行PCR扩增分析,其中13个含有肠毒素基因,为从分子水平探讨Bt与蜡质芽孢杆菌（Bacillus cereus,Bc)的肠毒素致病性差异奠定基础。