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PubMed2005

[The characteristics of staphylococcal enterotoxins produced by strains isolated from mastitic cows, including epidemiological aspects].

Staphylococcal enterotoxins (SEs) are big heterogenic group of exotoxins, rather differential in respect of their nucleotide and amino-acid homology, as well as the location of their genes, molecular weight and iso-electric point value. SEs were identified in 1959 as the extra-cellular proteins produced by some Staphylococcus aureus strains. These enterotoxins are known as the pyrogenic toxins and this group contains also other staphylococcal toxins (staphylococcal toxic-shock syndrome toxin--TSST-1, A and B exfoliative toxins and streptococcal scarlet fever toxin). Twenty one serological types of staphylococcal enterotoxins are distinguished. All of them are structurally and functionally similar to the toxic shock syndrome toxin (TSST-1). SEs are thermo-stabile proteins, resistant to many proteolytic enzymes (pepsin, trypsine, chymotrypsine, renine and papain), but this resistance depends on the temperature and pH. Staphylococcal enterotoxin-encoding genes are located as well in the chromosomal DNA, as on the pathogenicity island, in phages, transposones and plasmids. Enterotoxins are staphylococcal virulence factors responsible for food poisonings in humans. These proteins are also isolated from cows with mastitis. In various countries, the percentage of enterotoxin-producing S. aureus strains ranges from 5 to 60%, depending on the enterotoxine type. The variability and prevalence of enterotoxins produced by staphylococci isolated from mastitic cows is very important clinical and epidemiological problem. The analysis of enterotoxins interrelations, their structure, properties and occurrence, will provide better revealing their role in the emerging, development and spreading of human and animal diseases. Classical enterotoxins, as well as the new types of these proteins, are variable element of staphylococcal virulence that connects the occurence of mastitis with human food poisonings.

Adolescent↗

PubMed1989

Comparative structural analysis of staphylococcal enterotoxins A and E.

Structural analysis of staphylococcal enterotoxins A and E, two functionally and serologically related proteins, has been carried out using circular dichroism, and tryptophan fluorescence quantum yield and quenching. Secondary structures derived from the far-UV circular dichroic spectra revealed that both enterotoxins are in predominantly beta-sheets/beta-turn structures (80-85%). Staphylococcal enterotoxin A has significantly higher alpha-helical content (10.0%) than staphylococcal enterotoxin E (6.5%). Tryptophan fluorescence spectra of both enterotoxins showed maxima at approximately 342 nm, indicating that the fluorescent tryptophan residues are in polar environments. However, the tryptophan fluorescence quantum yields indicated that tryptophan residues are approximately 41% more fluorescent in staphylococcal enterotoxin A than in staphylococcal enterotoxin E. Tryptophan fluorescence quenching by a surface quencher, I-, and a neutral quencher, acrylamide, indicated that at least 1 of the 2 tryptophan residues in both staphylococcal enterotoxins A and E is located on the outer surface of the proteins. This tryptophan residue is in significantly different environments in the two enterotoxins. Six antigenic sites are predicted from the hydrophilicity and secondary structure information; at least four sites are identical. In general, staphylococcal enterotoxins A and E have some structural similarities which are compatible with their common biological activities.

Acrylamide↗

PubMed1988

Regulation of the enterotoxin B gene in Staphylococcus aureus.

The enterotoxin B gene of Staphylococcus aureus encodes a single mRNA of about 900 nucleotides. To identify the DNA sequences involved in transcription of the enterotoxin B gene, the transcription initiation and termination sites were determined by nuclease S1 protection experiments. Determination of the enterotoxin B mRNA and protein levels from a number of toxin-producing strains showed that strains that contained relatively low levels of mRNA synthesized low levels of enterotoxin B, whereas strains that carried high levels of enterotoxin B mRNA produced relatively high levels of the toxin protein. Strains carrying the cloned enterotoxin B gene secreted greatly reduced amounts of other extracellular proteins, indicating that the synthesis of several exoproteins in S. aureus is coordinately regulated. An accessory element called agr has been suggested to be involved in the regulation of several exoprotein genes in S. aureus. When the cloned enterotoxin B gene was introduced into strain ISP546 in which the agr element has been inactivated, reduced levels of both enterotoxin B and enterotoxin B mRNA were found. Our results suggest that the enterotoxin B gene is regulated at the transcriptional level and that the agr element plays a role in this regulation.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

Enterotoxin plasmids in bovine and porcine enterotoxigenic Escherichia coli of O groups 9, 20, 64 and 101.

Fourteen strains of Escherichia coli of serogroups characteristic of porcine class 2 enterotoxigenic E. coli isolated from pigs or calves were selected for genetic studies. The strains were examined for their ability to cotransfer a number of plasmid-mediated properties during conjugation with E. coli K-12. These properties were antibiotic resistance, and the production of heat-stable enterotoxin, the K99 antigen and colicin and the ability to ferment raffinose. Distinction was made between the two types of heat-stable enterotoxin, STa and STb. All 14 strains were antibiotic resistant and 11 of them cotransferred antibiotic resistance and heat-stable enterotoxin. One strain which transferred heat-stable enterotoxin also transferred the raffinose gene. Among six K99-positive strains which transferred heat-stable enterotoxin, five always cotransferred K99. Three strains had 100% cotransfer of colicin as well as heat-stable enterotoxin and K99. Drug resistance determinants were cotransferred at high frequency with heat-stable enterotoxin for six of eight multiple drug resistant enterotoxigenic E. coli. A 100% cotransfer of combinations of heat-stable enterotoxin, K99, colicin and antibiotic resistance was often associated with a single plasmid band on agarose gel electrophoresis. For some strains, the genes for STa and STb were on the same plasmid and for others they were on separate plasmids. The enterotoxin plasmids ranged in size from 5.2 to 85 Mdal. Heterogeneity in molecular size occurred among enterotoxin plasmids in E. coli of the same serogroup and recovered from the same animal host species.

Animals↗

PubMed1976

[Studies of enterotoxin production by strains of Staphylococcus aureus of different origins (author's transl)].

376 Staphylococcus aureus strains of clinical and epidemiological origin were tested for their ability to produce the enterotoxins, A, B and C1. For these investigations, microagargel precipitation against specific antisera has been used. Additionally these strains were studied by phagetyping, biochemical typing and for their antibiogram. The strains tested came from human pathological processes, mouth throat swabs of healthy people, persons working in hospitals, outbreaks of food poisoning from cows suffering from mastritis and some from milk samples. Most of the strains isolated from food poisonings were associated with enterotoxin-A-production. Among the group of strains from autopsy material, the enterotoxin-B-producing strains were predominant. The strains from human pathological processes which were found to be enterotoxin producing, were mostly belonging to phage group III. With regard to the antibiogram, the enterotoxin-producing strains were somewhat more resistent than the enterotoxin-negative strains. Correlations between the production of enterotoxin B and methicillin resistance did not become evident in our material. The strains were differentiated, too, as to their classification as var. hominis or var. bovis. With one exception, all enterotoxin-producing strains which could be classified in this way, were belonging to var. hominis. From five enterotoxin-producing strains isolated from cattle, only one strain belonged to var. bovis, the other to var. hominis. Obviously the ability to produce the enterotoxins, A, B and C1 is very rare in the group of var. bovis strains.

Animals↗

PubMed1978

Quantitative measurements of cholera enterotoxin in cultures of toxinogenic wild-type and nontoxinogenic mutant strains of Vibrio cholerae by using a sensitive and specific reversed passive hemagglutination assay for cholera enerotoxin.

A sensitive and specific reversed passive hemagglutination (RPHA) assay for cholera enterotoxin has been developed. Equine anti-choleragenoid antibodies purified by immunoadsorption were covalently coupled to formalinized sheep erythrocytes, using bis-diazotized benzidine, and the antitoxin-sensitized erythrocytes were shown to agglutinate specifically in the presence of cholera enterotoxin. In a microtiter RPHA assay system, the smallest quantity of enterotoxin that caused hemagglutination was approximately 20 pg. A sensitive assay for antibodies to enterotoxin was also developed, based on inhibition of RPHA. Using such assays, we demonstrated that several nontoxinogenic (tox-) strains of Vibrio cholerae produced small but detectable yields of enterotoxin, 4 to 16 ng/ml, under conditions where the highly toxinogenic strain 569B Inaba produced approximately 16 microgram of enterotoxin per ml. The enterotoxin produced in small quantities by these tox- strains was found to be identical to the enterotoxin from V. cholerae 569B Inaba iv its immunological and biological activities. Strains of V. cholerae that produce intermediate yields of enterotoxin have been obtained by two techniques: (i) as less toxinogenic mutants derived from highly toxinogenic strains and (ii) as more toxinogenic mutants derived from tox- strains. Thus, the yield of enterotoxin in cultures of V. cholerae grown under standardized conditions is is a genetically controlled trait that can be altered by mutation.

Antibody Specificity↗

PubMed1995

The enterotoxin of Bacteroides fragilis is a metalloprotease.

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.

Actins↗

PubMed1984

Comparative study of the nature and biological activities of bacterial enterotoxins.

It is apparent that there are considerable similarities between many of the enterotoxins produced by enteric pathogens. Although the effect of most of these toxins is restricted to the intestine in vivo, many cells are also sensitive to intoxication in vitro. The resultant in-vitro biochemical changes may have no pathological significance but serve to underline the central role of cyclic nucleotides in cellular fluid regulation. The biological activity of these enterotoxins is the result of interaction with membrane-bound adenylate cyclase, leading to persistent elevation of intracellular levels of cAMP. Stimulation of adenylate cyclase occurs consistently after a characteristic lag phase which varies somewhat between toxins. The duration and degree of stimulation of adenylate cyclase by the various toxins may point to possible differences in affinity, dissociation and mechanism of activation of the cyclase molecule. Subtle events at, or within, the cell membrane must occur during intoxication and may include complex associations of toxin with membrane lipid and protein components. The heat-labile toxins of V. cholerae, E. coli, Salmonella spp., A. hydrophila and Y. enterocolitica have much in common in their structures, membrane receptors and biochemical modes of action. Similarly the heat-stable toxins of E. coli and Y. enterocolitica, match each other in their biological activities. Classified along with the enterotoxin of C. perfringens, the enterotoxin produced by Sh. dysenteriae (and possibly some strains of E. coli) appears to differ from the other enterotoxins by acting on protein biosynthesis primarily and not on the nucleotide cyclase activation systems. In another category must be placed the various enterotoxins produced by Staph. aureus until more is known. Surprisingly little research has been directed towards the elucidation of their mode of action, although much is known of their serological and structural differences. Evidence to date suggests that staphylococcal enterotoxins differ from the other diarrhoeagenic agents discussed in this review. The structural and immunological similarities between the various heat-labile enterotoxins suggest a common genetic origin with gene transfer between the different bacterial species being responsible for the spread of enterotoxigenicity. It is possible that many of the "newer" enterotoxins owe their origin to genetic recombination with the "older" enteropathogens like V. cholerae.

Adenylyl Cyclases↗

PubMed1985

Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin.

Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin. Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution. All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains. These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue. The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA. Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule. The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay. Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin. Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes.

Animals↗

PubMed1989

Escherichia coli enterotoxin receptors: localization in opossum kidney, intestine, and testis.

The distribution of receptors for Escherichia coli enterotoxin were examined in opossum kidney, intestine, and testis. E. coli enterotoxin stimulated guanosine 3',5'-cyclic monophosphate (cGMP) production in renal cortex, testis, and small intestinal mucosa but had only a small effect in the colon. Atrial natriuretic factor enhanced the cGMP content of renal cortex and small intestine but had no effect on testis or colon. The enterotoxin receptors were observed to be localized in proximal tubules, to epithelial cells of crypts and villi of small intestine, to crypts of colon, and in seminiferous tubules. Both convoluted and straight portions of proximal tubules exhibited specific binding sites for 125I-labeled enterotoxin. Glomeruli and distal tubules did not have receptors. Binding of 125I-enterotoxin to brush-border membranes of kidney cortex or intestinal mucosa and to testis membranes was markedly temperature dependent. The binding affinities of these receptors for E. coli enterotoxin were similar (i.e., IC50 approximately equal to 0.4-0.5 nM). Daily administration of 20 micrograms of enterotoxin intramuscularly to opossums increased urine cGMP excretion with no apparent changes in urine volume, Na+, or K+ excretion. Thus receptors for heat-stable enterotoxins are localized to proximal tubules of kidney and to enterocytes and seminiferous tubules of intestine and testis, respectively. Apical membranes may be the site of enterotoxin receptors in these epithelia.

Animals↗

PubMed1980

The secondary structure of staphylococcal enterotoxins A, B and C.

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215--218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little alpha-helix as analyzed with CD models. A secondary structure of entertoxin B compured from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in alpha-helices, 71 in beta-pleated sheets, 88 in beta-turns and 55 in aperiodic conformation.

Chemical Phenomena↗

PubMed1987

Production and characterization of monoclonal antibodies to staphylococcal enterotoxins: use in immunodetection and immunopurification.

Four cell lines producing monoclonal antibodies were obtained by fusion of NS1 myeloma cells with splenocytes of BALB/C mice immunized with only 1 microgram of each staphylococcal enterotoxin A, B, C1 and D by a modified technique of intrasplenic boosting. This procedure was considerably more efficient than the more commonly used intravenous boosting. The antibodies EC-A1, EC-B1, EC-C1 and EC-D1, all of the IgG1 subclass, have high affinities for the corresponding enterotoxins A, B, C1 and D, with dissociation constants of 1.4, 2.8, 1.4 and 1.5 nM respectively; in addition EC-B1 showed a high affinity (2.1 nM) for enterotoxin C1. All these antibodies recognize, by immunoblotting, the homologous purified enterotoxins as well as enterotoxins from the bacterial culture supernatants. A rapid indirect double sandwich ELISA using a pair of antibody preparations was developed, where monospecific monoclonal antibodies were used to coat plastic plates and polyspecific rabbit antibodies were used to detect the enterotoxins under field conditions. These antibodies which are capable of immunoadsorbing the enterotoxins from staphylococcal culture filtrates and from natural fluids such as milk, were used to immunopurify enterotoxins A, C1 and D. The homogeneity and integrity of the affinity purified toxins A, C1 and D was verified by direct automated Edman degradation and yielded single amino terminal sequences which were moderately homologous to those published previously for B and C1 enterotoxins.

Animals↗

PubMed1975

Stimulation of cyclic adenosine 3':5'-monophosphate and corticosterone formation in isolated rat adrenal cells by cholera enterotoxin. Comparison with the effects of ACTH.

1. The production of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and corticosterone isolated ratadrenal cells was increased by cholera enterotoxin. Both responses were accompanied by a lag period which is characteristic of other known actions of enterotoxin. The duration of the lag period in the production of corticosterone depended on the concentration of enterotoxin; with the maximally stimulating amounts it was 30-45 min. 2. Maximum rates of cyclic AMP and corticosterone synthesis, after the lag period, were constant for at least 1 h. Although the maximum rate of corticosterone formation was the same as that obtained adrenocorticotropic hormone, the maximum rate of cyclic AMP formation was only 8-10% of that with adrenocorticotropic hormone. 3. Pretreatment of the cells with enterotoxin ahd no effect on their subsequent steroidogenic response to maximally stimulating amounts of adrenocorticotropic hormone. 4. Cycloheximide inhibited the effect of both enterotoxin and adrenocorticotropic hormone on corticosterone production. 5. Enterotoxin stimulation of both cyclic AMP and corticosterone formation was dependent on the presence of Ca2+ in the medium although the Ca2+ requirement was not same as that for adrenocorticotropic hormone. Thus, EGTA at concentrations which completely abolished the effect of adrenocorticotropic hormone caused only a partial reduction in the effects of enterotoxin. 6. Exogenously added choleragenoid and gangliosides abolished the effects of enterotoxin without having any significant effect on the response of the cells to adrenocorticotropic hormone. 7. After treatment with neuraminidase, the adrenal cells showed an increased response to enterotoxin in terms of both cyclic AMP and corticosterone formation which was due to a combination of two effects: (a) increased rate of synthesis of both compounds and (b) shortening of the characteristic lag period. This is in sharp contrast to the results obtained with adrenocorticotropic hormone where neuraminidase-treatment made the cells less sensitive to adrenocorticotropic hormone.

Adrenal Glands↗

PubMed1986

Detection of Clostridium perfringens enterotoxin in stool specimens and culture supernatants by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to detect and quantitate Clostridium perfringens enterotoxin A in culture supernatants and in stool specimens from cases of diarrhea in which high numbers of enterotoxin-producing Clostridium perfringens were isolated. To analyze for enterotoxin A, polyvinyl chloride microtiter plates were coated with dilute immune whole rabbit serum. Enterotoxin A standards and samples were allowed to react with sensitized wells. The presence of the immobilized antigen in the wells was detected by the binding of immune rabbit immunoglobulin conjugated with peroxidase. Nanograms of enterotoxin were detectable. Four enterotoxin-positive and seven enterotoxin-negative cultures grown in Duncan-Strong medium gave expected results. Eighteen of 23 diarrheal stool specimens obtained after a food-poisoning outbreak at a state hospital were found to contain microgram quantities of enterotoxin per gram of stool, whereas five control diarrheal specimens contained less than 0.6 ng enterotoxin per gram of stool. These results indicate that the enzyme-linked immunosorbent assay technique is useful for differentiating enterotoxigenic strains and for diagnosing diarrhea caused by enterotoxigenic Clostridium perfringens.

Antibodies, Bacterial↗

PubMed2000

Involvement of kinins, mast cells and sensory neurons in the plasma exudation and paw oedema induced by staphylococcal enterotoxin B in the mouse.

Intraplantar injection of staphylococcal enterotoxin B induces long-lasting oedema mediated by both cyclooxygenase and lipoxygenase products as well as by neuropeptides from sensory nerves. This study was undertaken to further clarify the role of peripheral primary afferent sensory nerves in staphylococcal enterotoxin B (25 microg/paw)-induced plasma extravasation and oedema formation. The tachykinin NK(1) receptor antagonist (S)-1-[2-[3-(3, 4-dichlorophenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azoniabicyclo [2.2.2]octane cloride (SR140333; 120 nmol/kg, s.c.+120 nmol/kg, i.v.) significantly inhibited plasma exudation and paw oedema evoked by staphylococcal enterotoxin B. The tachykinin NK(2) receptor antagonist (S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3, 4-dichlorophenyl)butyl]-benzamide (SR48968) had no effect on the staphylococcal enterotoxin B-induced responses. The bradykinin B(2) receptor antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]bradykinin (Hoe 140; 400 nmol/kg, i.v.) significantly reduced staphylococcal enterotoxin B-induced responses. The magnitude of the inhibition observed with Hoe 140 alone was similar to that caused by concomitant treatment of animals with SR140333 and Hoe 140, suggesting that there is a final common pathway. Additionally, SR140333 given alone reduced bradykinin (3 nmol/paw)-induced paw oedema. The vanilloid receptor antagonist N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7, 8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine; 100 micromol/kg) significantly reduced staphylococcal enterotoxin B-induced responses. The 5-HT receptor antagonist methysergide (10 mg/kg, i.v.) and the histamine H(1) receptor antagonist mepyramine (10 mg/kg, i.v.) produced a significant reduction in paw oedema whereas plasma exudation was reduced only by methysergide. In diabetic mice, exudation and oedema evoked by staphylococcal enterotoxin B were markedly reduced. Acute administration of insulin (20 UI/kg, s.c., 30 min before) did not restore the increased permeability induced by staphylococcal enterotoxin B. We conclude that plasma exudation and paw oedema in response to staphylococcal enterotoxin B are a consequence of a complex neurogenic response involving direct activation of vanilloid receptors on sensory nerves, release of kinins and subsequent activation of bradykinin B(2) receptors at a prejunctional level, and direct or indirect degranulation of mast cells.

Animals↗

PubMed1980

Binding of Clostridium perfringens [125I]enterotoxin to rabbit intestinal cells.

125I-Labeled enterotoxin from Clostridium perfringens was utilized to characterize the association of the enterotoxin with cells isolated from rabbit intestine and tissue homogenates from liver, kidney, and brain. The enterotoxin was found to bind in a specific and saturable manner to cells from intestine and to tissue homogenates from liver and kidney but not the brain. Detailed studies of the binding were carried out with the ileal epithelial intestinal cells. The rate and amount of binding of enterotoxin to cells appeared to be temperature dependent. Apparent affinity and association and dissociation rate constants were calculated for what appeared to be two classes of saturable binding sites. The amount of enterotoxin molecules that bound per milligram of cell protein was similar in tissue of intestinal, liver, and kidney origin (approximately 10(13) molecules/mg of cell protein). Spontaneous dissociation into the supernatant medium was observed to be much slower than expected from calculations based on the rate of association. Chaotropic ions did not enhance dissociation of the enterotoxin from cells. Enterotoxin binding was demonstrated to be heat labile (binding ability was lost after the enterotoxin was heated for 10 min at 60 degrees C). A mechanism is described whereby the enterotoxin binds and then is inserted into the membrane where it becomes trapped.

Animals↗

PubMed1997

Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin.

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and pore formation in the cell membrane.

Amino Acid Sequence↗

PubMed1996

Serum antibody response to Staphylococcus aureus enterotoxins and TSST-1 in patients with septicaemia.

The prevalence of enterotoxins and toxic shock syndrome toxin (TSST-1) production in strains isolated from patients with Staphylococcus aureus septicaemia, and the serum antibody response in relation to toxin production in vitro of each isolate, were investigated. Among 63 strains of S. aureus isolated from the blood of patients with septicaemia, 51 from patients with superficial wounds and 49 from nasal carriers, 50-60% produced at least one of the enterotoxins A-D or TSST-1. The most frequent toxins produced were enterotoxins A and C and TSST-1. Among the 63 patients with staphylococcal septicaemia, 51 (81%) had a significant rise or a high antibody titre, or both, to at least one of the toxins. A positive serological response to toxin A was found in 78%, to enterotoxin B in 83%, to enterotoxin C in 80%, to enterotoxin D in 86% and to TSST-1 in 92% of the patients from whom the isolated strain produced the respective toxin. Antibodies against enterotoxins A, B, C and D and TSST-1 were also seen in 35%, 16%, 32%, 59% and 10%, respectively, in patients infected by strains that did not produce the specific toxin. Immunological cross-reactions between the toxins were demonstrated both in hyperimmune sera obtained from rabbits and in patients' sera, particularly between enterotoxins B and C. It is concluded that these potent toxins with superantigenic properties are produced in vivo during S. aureus septicaemia. No differences with regard to enterotoxin or TSST-1 production or antibody response were noted between patients with complicated versus uncomplicated septicaemia.

Adolescent↗