Properties of homogeneous heat-labile enterotoxin from Escherichia coli.

Recently, the heat-labile enterotoxin (LT) of Escherichia coli has been purified to homogeneity and partially characterized (Clements and Finkelstein, Infect. Immun. 24:760-769, 1979). This study extends our observations on the physicochemical properties of LT. The toxin has an isoelectric point of pH 8.0, as compared with choleragen and choleragenoid, which have isoelectric points of pH 6.75 and 7.75, respectively. Sedimentation equilibrium measurements established an approximate molecular weight for LT of 91,440. LT had an even more marked affinity than choleragen for agarose-containing matrixes in gel filtration. Of several mono- and disaccharides tested, only galactose and lactose were highly efficient in removing 125I-labeled LT from agarose-containing columns. LT dissociated into subunits (designated A and B) during gel filtration in the presence of 5 M guanidine. These subunits were immunologically distinct and possessed unique and shared antigenic determinants to the corresponding A and B subunits of choleragen. During gel filtration of LT at pH 6.5 and room temperature, a spontaneously occurring toxoid of LT, analogous to choleragenoid, was discovered and designated "coligenoid." This product contains only the B subunits of the toxin. A partial amino acid sequence of the B subunit of LT revealed a remarkable homology to the primary structure of cholera toxin B. Within the first 20 amino acids of the two chains, only 5 differ, and these differences may be attributable to single base substitutions.     

Cellular release of heat-labile enterotoxin of Escherichia coli by bacteriophage induction.

Treatment of some enterotoxigenic Escherichia coli strains with the antibiotic mitomycin C resulted in lysis of the bacteria. Heat-labile enterotoxin (LT) activity of culture filtrates, determined by means of the Y-1 adrenal cell assay, increased dramatically as lysis of the culture proceeded. Further studies with E. coli strains 263 and B21-4 revealed that lysis is due to mitomycin C induction of vetetative development of a temperature bacteriophage. These findings suggest that the elevated levels of LT detected after mitomycin C treatment reflect the lytic release of cell-bound LT rather than the induction by mitomycin C of de novo toxin biosynthesis. Comparable increases in LT activity also resulted from thermal induction of a phage P1Cm lysogen of strain 263 or from sonic disruption of enterotoxigenic strains.     

Factors influencing heat-labile Escherichia coli enterotoxin activity.

In this study, conditions for production, detection, and storage of heat-labile Escherichia coli enterotoxin (LT) in culture filtrates from E. coli H-10407 were defined by using the adrenal tumor cell assay system. An enriched medium containing 0.6% yeast extract, 2% Casamino Acids, and 0.25% glucose buffered at pH 8.5 produced the highest LT activity of the various test media. In E. coli strain H-10407, LT activity was markedly decreased if the initial pH of the culture media was reduced to pH 7.5 or less. In contrast to E. coli P-263, if strain H-10407 was grown in the presence of mitomycin C there was no increase in LT production. Crude-culture filtrates containing LT can be stored at 4 degrees C for several days without an appreciable loss of activity; however, for long-term storage lyophilization or freezing at -70 degrees C is recommended.     

Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity.

The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2. This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity. Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone. Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice.     

Zinc and the heat-labile enterotoxin of Escherichia coli

Enterotoxigenic Escherichia coli is a major cause of diarrhoea in man. When zinc in concentrations of 10(-6) M or 10(-5) M was added to the growth medium, there was a significant increase in heat-labile enterotoxin production by each of six toxigenic strains. Zinc in these concentrations did not alter bacterial growth or the activity of preformed toxin. Other heavy metals did not enhance toxin production and o-phenanthroline, a relatively specific zinc-chelating compound, blocked the enhancing effect. The significance of these findings is discussed in relation to the use of supplemental dietary zinc.     

Assay of Escherichia coli heat-labile enterotoxin with vero cells.

The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains of Escherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory.     

Subunit number and arrangement in Escherichia coli heat-labile enterotoxin.

The Escherichia coli heat-labile enterotoxin (LT) was found to have the same subunit structure as cholera toxin, namely, one A subunit and five B subunits. Reaction with a bisimidate generated all the possible cross-linked derivatives of A5B: B,2B ... 5B and A, AB ... A5B. The isolated B component, coligenoid, contained five B subunits and showed some tendency of polymerize: with a bisimidate it became covalently connected into the set B ... 5B with lesser amounts of 6B ... 10B, etc. The subunit formulas of two independently prepared samples of LT were both proved to be A5B by cross-linking, but their B pentamers migrated at different rates on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that they have different conformations. The faster (R) form could be converted to a diffuse slower (C) form by incubating it at 50 degrees C or at 37 degrees C with 0.2 M galactose, which is the terminal sugar of ganglioside GM1, the natural receptor for LT. Cholera toxin resembled the R form more than the C form of LT.     

Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.     

Microtiter solid-phase radioimmunoassay for detection of Escherichia coli heat-labile enterotoxin.

The development of a microtiter solid-phase radioimmunoassay for the detection of Vibrio cholerae enterotoxin and heat-labile Escherichia coli enterotoxin is described. The test is based on the immunological similarity between V. cholerae toxin and E. coli heat-labile toxin. The assay is easy to perform, quantitative, and at least as sensitive and specific as the Y-1 adrenal cell system.     

Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin.

The antigenic relationships between heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (strain 286C2) and cholera toxin (CT) were examined by using antisera raised against LT and CT and specific antisera prepared against each subunit of both enterotoxins. Double immunodiffusion analysis revealed reactions of partial identity between the A subunits of LT and CT, as well as between the B subunits. Rabbit antisera raised against LT subunit A reacted with only subunit A, whereas rabbits immunized with LT subunit B produced antibodies which reacted with only subunit B. A high degree of CT neutralization was observed with antisera raised against LT. Data from neutralization assays with specific antisera to each enterotoxin showed that LT was more effectively neutralized by homologous anti-LT than CT (3.7-fold); however, anti-CT was only slightly more effective in neutralization of homologous CT compared with LT (1.9-fold). In contrast, antisera raised against the B subunit of CT (choleragenoid) exhibited significantly higher neutralization activity against CT than LT (5.8-fold); however, the amount of CT neutralized by anticholeragenoid was less (4.1-fold) than anti-CT. These results suggested that anti-CT serum contained neutralizing antibodies reactive with a shared determinant formed by interaction of the A and B subunits, whereas anti-LT and anti-choleragenoid sera did not. Sensitive solid-phase radioimmunoassays were developed to examine the affinity and degree of specificity involved in homologous and heterologous antigen-antibody interactions between LT, CT, their subunits, and specific antibodies. Only unlabeled LT competed with radiolabeled LT in polystyrene tubes coated with anti-LT, and only unlabeled CT competed with radiolabeled CT in tubes coated with anti-CT. However, when radiolabeled CT was incubated in tubes coated with anti-LT, competitive inhibition responses were observed with both unlabeled toxins. When radiolabeled LT was incubated with tubes coated with anti-CT, competitive inhibition responses were observed with both unlabeled toxins. Similar competitive inhibition responses were observed with the A subunits of LT and CT and with the B subunits using antisera specific for the subunits of each enterotoxin. Double immunodiffusion analysis and radioimmunoassay data supported the presence of unique and shared immunodeterminants in each subunit.     

Assay for heat-labile Escherichia coli enterotoxin using sandwich erythroimmunoassay

We investigated the possibility of using a sheep erythrocyte-antibody conjugate as reagent in a sandwich erythroimmunoassay (SERIA) procedure to detect and titrate Escherichia coli heat-labile enterotoxin (LT) with the naked eye. In this assay, which is based on the immunological similarity between Vibrio cholerae toxin (CT) and LT, rabbit anti-CT IgG was used as immunosorbent, and sheep erythrocytes, sensitized with the rabbit anti-CT antibodies, were used as indicator. The sensitivity of the test was demonstrated by a comparative study using an enzyme-linked immunosorbent assay (ELISA). The results obtained by SERIA with 130 samples correlated well with those of a Vero cell assay and a GM₁-ELISA. The test is easy, relatively cheap and as sensitive as other standard techniques; it is particularly suited for field laboratories, especially in tropical countries, and a large number of strains may be examined daily.     

Purification and characterization of type II heat-labile enterotoxin of Escherichia coli.

Type II heat-labile enterotoxin (LT-II) of Escherichia coli has several biologic activities similar to cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LT-I), but it is not neutralized by antiserum prepared against CT or LT-I. LT-II was purified from E. coli SA53 and from E. coli HB101(pCP3837), a strain that contains the cloned LT-II genes in a hybrid plasmid and produces up to 600 times more LT-II than does SA53. Purification involved sonic disruption of bacterial cells, ammonium sulfate fractionation, chromatography on Affi-Gel Blue, chromatofocusing, and gel filtration on Sephadex G-100. The LT-II purified to apparent homogeneity from HB101(pCP3837) had an isoelectric point of 6.8, induced increased vascular permeability in rabbit intracutaneous tests, caused rounding of cultured Y1 adrenal cells accompanied by increased intracellular cyclic AMP, and was 25 to 50 times more potent than CT or LT-I in the Y1 adrenal-cell assay. In contrast, purified LT-II did not cause secretion in ligated rabbit ileal segments at doses corresponding to CT controls that gave strongly positive reactions. LT-II was composed of two different polypeptides with MrS of 28,000 (A) and 11,800 (B); treatment of LT-II with trypsin cleaved the A polypeptide to fragments A1 (Mr, 21,000) and A2 (Mr, 7,000). The activity of LT-II was not blocked by ganglioside GM1 at concentrations that inactivated LT-I or CT. Antiserum against the LT-II from E. coli HB101(pCP3837) completely neutralized purified LT-II and the LT-II in crude extracts of SA53, but it did not neutralize purified LT-I or CT.     

Partial purification and characterization of a heat-labile enterotoxin of Escherichia coli.

A partially purified enterotoxin was obtained from the growth medium of Escherichia coli strain 711 (P307), a derivative of E. coli K-12, by ultrafiltration, precipitation with ammonium sulfate, molecular sieving, and anion exchange column chromatography. The active moiety, which is heat-labile, behaved like a protein particle of 180,000 to 200,000 daltons during molecular sieving and ultracentrifugation. During polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), it dissociated into two subunits with apparent molecular weights of 68,000 to 70,000 and 14,000 to 15,000. SDS-PAGE after heating in SDS changed the larger subunit to an apparent molecular weight of about 40,000; the smaller subunit did not change. The intact particle induced rounding of the cells in Y-1 mouse adrenal tumor cells used for assay. The detergent-dissociated molecules were not active. Proteolysis of the purified toxin by tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin appeared to enhance its activity. The addition of serum to the assay medium resulted in partial depression of the activity. Activity was also abolished by preincubation of the toxin with either a rabbit antiserum to it or solutions containing GM1 ganglioside. The length of time needed to evoke a response in the assay system by fractions from different stages in the purification of the enterotoxin was a useful parameter in the evaluation of specific activity.     

Assay of the heat-labile enterotoxin of Escherichia coli in infant rabbits.

Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing. Infant rabbits could be used in a simple quantitative assay for heat-labile E. coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing. Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin. Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin. Lincomycin andmitomycin C were found not to increase th e yield of heat-labile enterotoxin from E. coli strain P307.     

Comparison of preservation methods for enterotoxigenic Escherichia coli producing heat-labile enterotoxin.

Ten strains of enterotoxigenic Escherichia coli producing heat-labile enterotoxin (LT) were preserved under 12 different conditions. After 1 month, 9 months, and 3 years of preservation, the cultures were recovered and examined for LT production. Preservation of the cultures on Dorset Egg Medium at 4 degrees C and preservation by freezing the cell suspensions in tryptic soy broth with 20% glycerol were found to be suitable preservation methods; all strains were alive for 3 years and had a minimum loss of LT production.     

Induction of heat-labile enterotoxin synthesis in enterotoxigenic Escherichia coli mitomycin C.

Introduction of heat-labile toxin (LT) synthesis in enterotocigenic strains of Escherichia coli by mitomycin C (MTC) was demonstrated. Six enteropathogenic strains which produce LT were inducible, exhibiting an 896-fold increase in LT when compared to ininduced cultures. On the other hand, four nonenteropathogens and three other pathogens which produce only the heat-stable toxin were not induced to produce LT. Gel filtration chromatography, antibody neutration, and heat lability studies suggest that the toxin synthesized in the presence of MTC is the same as the toxin synthesized in the absence of MTC.     

Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli.

Reactivity to Trypanosoma cruzi antigens and autoreactivity to heart antigens were evaluated in 27 patients with Chagasic cardiomyopathy (group I), 52 patients without evidence of cardiac dysfunction (group II), and 36 selected controls, either healthy patients or patients with other heart diseases (group III). The in vitro lymphoblastogenesis response to T. cruzi antigens was found to be high in groups I and II and low in group III. The mean stimulation index to T. cruzi antigens, in fact, tended to be highest in group I, suggesting a more intense immune response in patients with Chagasic cardiomyopathy. The proportion of individuals with reactivity to heart antigens was 28.6% in group I, 25% in group II, and 0% in group III. The finding of an equal percentage of reactivity to heart antigens in groups I and II was unexpected, as a higher incidence of positive reactions in group I was predicted. Consequently, it is thought that this finding and its relevance to the pathogenic process of Chagasic cardiomyopathy need to be carefully assessed in a longitudinal study.     

Intracellular distribution of heat-labile enterotoxin in a clinical isolate of Escherichia coli.

The intracellular distribution of heat-labile enterotoxin in a human isolate of enterotoxigenic Escherichia coli varied significantly as a result of changing incubation time, media, and degree of aeration. Direct comparison with a K-12 plasmid recipient revealed a similar but less dramatic response to environmental factors.     

GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin.

A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoassay was somewhat less sensitive than the GM1 enzyme-linked immunosorbent assay but more sensitive than the Vero cell assay. Results obtained for 12 LT-positive and 138 LT-negative E. coli strains correlated with results obtained with GM1 enzyme-linked immunosorbent and Vero cell assays.     

Comparison of five assays for the heat-labile enterotoxin of Escherichia coli

Enzyme-linked immunosorbent assay (ELISA), DNA-DNA hybridisation, Vero cell assay, the Biken test and a new membrane-filter method were compared in the detection of heat-labile enterotoxin (LT) of Escherichia coli. Six subcultures of each of 50 strains of E. coli from the Biken collection were evaluated "blind" in the laboratory. The combined results of the most reproducible tests (ELISA and DNA-DNA hybridisation) were used to calculate the sensitivity and specificity of the other assays. The Vero-cell assay had a high sensitivity (98%) but a lower specificity (91%). The Biken and membrane-filter assays had sensitivities of 58-71% and 77-84% respectively, depending on the type of antiserum used. Only one false positive result was obtained with the Biken test; specificity of the membrane-filter assay was 94-95%. The membrane-filter assay, with anti-cholera toxin, is specific and reasonably sensitive. It has particular advantages over DNA-DNA hybridisation and the Biken test, and it may prove suitable for screening large numbers of E. coli isolates in epidemiological studies in developing countries.