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We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.
The nucleotide sequence of the DNA encoding the ADP-ribosyltransferase (A1) fragment of cholera enterotoxin was determined. A putative precursor of the A1 peptide contains an 18-amino acid leader peptide, and the mature A1 peptide contains 194 amino acids. The primary structure of the A1 fragment from cholera enterotoxin is more related to that from a human enterotoxigenic Escherichia coli than to that from a porcine enterotoxigenic E. coli.
The gene (entD) encoding staphylococcal enterotoxin D (SED) has been located on a 27.6-kilobase penicillinase plasmid designated pIB485. This plasmid was present in all SED-producing strains tested. The entD gene was cloned on a 2.0-kilobase DNA fragment and was expressed in Escherichia coli. Sequence analysis of this fragment revealed an open reading frame that encoded a 258-amino-acid protein that possessed a 30-amino-acid signal peptide. The 228-amino-acid mature polypeptide had a molecular weight of 26,360 and contained a high degree of sequence similarity to the other staphylococcal enterotoxins. S1 nuclease mapping showed that transcription of entD was initiated 266 nucleotides upstream from the translation start codon. The entD gene was also shown to be activated by the staphylococcal regulatory element known as agr.
Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringens enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpe promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of cpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for beta-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpe promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.
The quorum-sensing system of Staphylococcus aureus, the accessory gene regulator (Agr) system, is responsible for increased transcription of certain exoprotein genes and decreased transcription of certain cell wall-associated proteins during the postexponential phase of growth. This regulation is important for virulence, as evidenced by a reduction in virulence associated with a loss of the Agr system. The enterotoxin D (sed) determinant is upregulated by the Agr system. To define the Agr-regulated cis element(s) within the sed promoter region, we utilized promoters not regulated by Agr to create hybrid promoters. Hybrid promoters were created by using sed sequences combined with the enterotoxin A (sea) promoter or the S. aureus lac operon promoter sequences. The results obtained indicated that the Agr control element of the sed promoter resides within the -35 promoter element and at the Pribnow box to the +1 site of the promoter. At these positions of the sed promoter, a directly repeated 6-bp sequence was found. This repeat is important for overall promoter activity, and maximal regulation of the promoter activity requires both repeat elements. Furthermore, Agr control of sed promoter activity was found to be dependent upon the presence of a functional Rot protein. Therefore, the postexponential increase in sed transcription results from the Agr-mediated reduction in Rot activity rather than as a direct effect of the Agr system.
The accessory gene regulator (Agr) system is a quorum-sensing system of Staphylococcus aureus responsible for upregulation of certain exoprotein genes and downregulation of certain cell-wall associated proteins during the post-exponential phase of growth. The enterotoxin B (seb) determinant is upregulated by the Agr system. Agr-regulated cis elements within the seb promoter region were examined by deletion analyses of the seb promoter by a hybrid promoter approach utilizing the staphylococcal lac operon promoter. To identify the regulatory pathway for enterotoxin B expression, the seb promoter fused to the chloramphenicol acetyltransferase reporter gene was introduced into mutants of S. aureus lacking agr or different members of the Sar family of transcriptional regulators. Agr control of seb promoter activity was found to be dependent upon the presence of a functional Rot protein, and Rot was shown to be able to bind to the seb promoter. Therefore, the Agr-mediated post-exponential-phase increase in seb transcription results from the Agr system's inactivation of Rot repressor activity.
Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.
The inhibitory effect of trypsin on the cytotoxicity of staphylococcal enterotoxin B acting with human embryonic intestine cell cultures was examined. Trypsin treatment of the cells rendered them resistant to enterotoxin for a period of 48 hr. The resistance increased proportionally with increased time of exposure of the cells to trypsin. Neither ethylenediaminetetraacetic acid nor scraping, which were used as alternate means of cell suspension, caused any resistance to the toxin. The effect is enzymatic and appears to be similar to the inhibitory action of trypsin and chymotrypsin on the attachment of polioviruses and coxsackieviruses to HeLa cells.
Enterotoxin B formation by Staphylococcus aureus S6 was inhibited by Tween 80, oleic acid, sodium deoxycholate, penicillin, d-cycloserine, or bacitracin. Toxin formation by strain 243 was sensitive to oleic acid, sodium deoxycholate, sodium lauryl sulfate, d-cycloserine, or bacitracin. The effect of d-cycloserine was reversed by d-alanine with strain 243 but not with strain S6. Neither penicillin nor bacitracin inhibited alpha-hemolysin or coagulase activity of strain S6; however, 0.118 mumoles of d-cycloserine per ml increased the alpha-hemolysin titer more than eightfold. Pigmentation of strain 243 was reduced by oleic acid, sodium deoxycholate, or methicillin, and was completely inhibited by d-cycloserine or bacitracin. Glucose was required for the inhibition by spermine of (14)C-valine incorporation into cellular protein of strain S6. These data indicate that the cell surface may contain sites important to the synthesis of enterotoxin B.
We investigated the effect of time, temperature, and the presence of sodium chloride, nitrates, and nitrites in the medium on the growth and production of enterotoxin B by Staphylococcus aureus. Assays by the double gel-diffusion method showed that maximal enterotoxin B production occurs at the beginning of the stationary phase of growth. Lowering the temperature of incubation decreased the amount of toxin produced without affecting the total amount of growth. Increases in concentration of curing salts reduced toxin production more rapidly than cell growth. The relationship of these observations to food-poisoning outbreaks is briefly discussed.
Monkeys were immunized with enterotoxin and enterotoxoid by intracutaneous injection or by feeding. Identical schedules were used to compare the effectiveness of the two antigens and the two routes. Enterotoxin administered intracutaneously was the most effective antigen, whereas oral administration of enterotoxoid was least effective. Intracutaneous injection of toxoid and oral feeding of toxin were intermediate and not too dissimilar in effectiveness. Antibody titers and protection persisted for at least 1 year at a relatively high level. Monkeys that had preimmunization hemagglutinins showed an anamnestic response after immunization. The development of protection and the appearance of antibodies subsequent to feeding toxin or toxoid suggest that ingestion of food contaminated by staphylococci or their metabolites may be one cause for the appearance of antitoxin in the serum of supposedly unexposed animals and man.
The Elek test was modified for detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli. A total of 164 strains of E. coli were tested by the modified Elek test, and the results correlated well with those of the Chinese hamster ovary cell assay and passive immune hemolysis. It is concluded that the modified Elek test is a simple and reproducible assay method for identification of E. coli which produce heat-labile enterotoxin, and is suitable for use in clinical laboratories.
A single radial immune hemolysis test for the detection of thermolabile enterotoxin has been developed for routine purposes. Stationary cultures from enterotoxigenic Escherichia coli in Casamino Acids-yeast extract medium may be used for the detection of this enterotoxin, and under the conditions of the experiment, the single radial immune hemolysis test was as sensitive as the passive immune hemolysis test. The results obtained in the single radial immune hemolysis test agreed entirely with those obtained in the passive immune hemolysis test, and no false-positive reactions were obtained when cholera antitoxin diluted 1:80 was used. The assay is easy to perform, inexpensive, and specially designed for less-equipped laboratories.
Milk specimens, 75 from cows immunized against cholera toxin and 35 from a human population in which enterotoxigenic Escherichia coli and rotaviral infections are endemic, were collected as paired filter paper and frozen whole milk samples. Each pair was tested for antibody activity against heat-labile E. coli and Vibrio cholerae enterotoxins. Additionally, 12 of the 35 paired human milk samples stored as frozen whole milk and dried on filter paper were tested for anti-rotavirus immunoglobulin A. Anti-enterotoxin and anti-rotavirus immunoglobulin A titers in milk dried on filter paper compared favorably with those of their frozen whole milk pairs. Filter paper samples offered considerable advantages for field collection, transportation, and storage over frozen liquid samples.
A solid-phase sandwich assay that was able to differentiate heat-labile-enterotoxin-producing colonies of Escherichia coli and choleratoxin-producing colonies of Vibrio cholerae from nontoxigenic colonies is described. Flexible polyvinyl chloride plastic film coated with antibody molecules was allowed to react with partially lysed bacterial colonies in a standard petri dish. The immobilized antigen on the plastic film was then labeled with radioiodinated antibody. Autoradiography identified antigen-containing colonies. As little as 5 to 25 pg of pure toxin contained in a 3- to 4-mm-diameter circle was reliably detected by this method. The synthesis of heat-labile enterotoxin and choleratoxin by cells growing on selective media such as eosin methylene blue agar, MacConkey agar, Endo agar, and thiosulfate-citrate-bile salts-sucrose agar was demonstrated. The method appears to be suitable for large-scale surveys.
We compared four versions of the enzyme-linked immunosorbent assay for their suitability for detecting staphylococcal enterotoxins. The sandwich with labeled antibody proved to be the best. We used it with a sorbent consisting of antibody-coated polystyrene spheres reacted with 20 ml of food extract. The sensitivity of the test was 0.1 ng of enterotoxin per ml, which is far below clinical relevance. The succinimidyl-pyridyl-dithio-propionate enzyme coupling method of Pharmacia was superior to the two-step glutaraldehyde technique. Interfering protein A was eliminated by the simple addition of normal rabbit serum to the extracts. A diagnostic kit is now available.
Three simple immunological tests, the modified Elek (Biken) test, the modified staphylococcal coagglutination test, and the rapid GM1-horseradish peroxidase-enzyme-linked immunosorbent assay have been evaluated for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. Of the 100 coded E. coli strains tested, 94 gave consistent results with all the three immunological tests; a discrepancy was observed in only 6 strains. Identical results were obtained when the Biken test was conducted with complete and incomplete Biken kits (Meguro Institute Ltd., Osaka, Japan). All three immunological tests evaluated in this study were found to be sensitive and simple and can be easily adopted by any laboratory for detection of heat-labile enterotoxins of enterotoxigenic E. coli strains.
Serum immunoglobulin G antibodies to purified heat-labile enterotoxin (LT) from human (LTh) and porcine (LTp) Escherichia coli strains and cholera enterotoxin (CT) were measured by an enzyme-linked immunosorbent assay. Sera from patients with LTh E. coli infection showed a prominent response with LTh, an intermediate response with LTp, and a meager response with CT. Of 47 persons with clinical LTh-producing E. coli (herein shortened to LTh E. coli) infections, significant rises in antitoxin were detected against LTh in 36 (77%), against LTp in 30 (64%), and against CT in only 13 (28%) patients; seroconversions also occurred in 11 of 14 (79%) patients with subclinical LTh E. coli infections. In North Americans with experimental LTh E. coli infection, anti-Lth did not remain at high levels for more than 3 months. Persons with cholera manifested antitoxin responses that were similarly potent against all three toxin antigens; in fact, net optical density values were often slightly higher against LTh than against CT. The ratio of CT/LTh ELISA net optical density in convalescent sera proved to be a sensitive means to differentiate LT E. coli from cholera infection. All 11 cholera patients tested had CT/LTh ratios of greater than 0.70, whereas in only 1 of 47 LTh E. coli infections did the ratio exceed that value (it was 0.71) (P less than 0.0000000001). In single serum specimens, a net optical density of greater than or equal to 0.30 against LTh was shown to be a useful cutoff in screening sera for recent LTh E. coli or past cholera infection. The CT/LTh ratio was then used to differentiate definitively. Sera from healthy 3- to 5-year -olds and 15- to 19-year-olds in Maryland, Chile, and Bangladesh were tested against LTh and CT. The serological results fit known epidemiological observations. (i) LTh infections are rare in the United States (only 2 of 60 sera had LTh net optical density values of >/= 0.30. (ii) In contrast, evidence of recent LTh E. coli infections was very common in Chilean (69%) and Bangladeshi (57%) 3- to 5-year-olds and not uncommon in 15- to 19-year-olds (38 and 31%, respectively) in those countries. (iii) Only Bangladeshi sera showed serological evidence of cholera infections (CT/LTh ratios of > 0.70). The immunoglobulin G enzyme-linked immunosorbent assay measuring antibodies to purified LTh and CT represents a practical and effective tool for the serological study of LTh E. coli and cholera diarrheal infections.