A new heat-labile cytolethal distending toxin (CLDT) produced by Escherichia coli isolates from clinical material

A new heat-labile Escherichia coli toxin cytolethal to Vero, HeLa, HEp-2 and CHO cells and negative in Y-1 cells has been demonstrated in culture filtrates of 43 E. coli strains associated with diarrheal disease. This new toxin was termed a cytolethal distending toxin (CLDT) to reflect the progressive cell distention and cytotoxicity evidenced in all sensitive tissue cells. CLDT was distinct from the classic heat-labile (LT) and heat-stable enterotoxins, Verotoxins and hemolysins and was produced by some strains of the following E. coli serogroups (02, 07, 08, 018, 022, 039, 044, 055, 083, 086, 091, 0113, 0119, 0128 and 0167). Moderate cyclic AMP accumulation (75-fold) was observed in CHO cells exposed to E. coli CLDT for 24 h. In contrast to E. coli LT, cyclic AMP levels were optimal at 24 h and were observed to decrease over the following 72 h period in CHO cells exposed to E. coli CLDT. In addition to heat-lability at 70 degrees C for 15 min, E. coli CLDT demonstrated a molecular weight over 30,000, was nondialyzable and trypsin-sensitive. E. coli CLDT was negative in adult rabbit ligated ileal loop and suckling mouse assays and provoked only an erythematous response in rabbit skin. All CLDT-positive E. coli strains identified to date were non-hemolytic and non-invasive. Verotoxin and heat-labile enterotoxin were found in combination with CLDT in a few E. coli strains.     

Comparison of the Action of Escherichia coli Enterotoxin on the Thymocyte Adenylate Cyclase-Cyclic Adenosine Monophosphate System to That of Cholera Toxin and Prostaglandin E1

Mouse thymocytes were used to compare mechanisms by which Vibrio cholerae and heat-labile Escherichia coli enterotoxins activate the adenylate cyclase-cyclic adenosine monophosphate (AMP) system. Both enterotoxins had their time-delayed increase in cyclic AMP neutralized by antisera to V. cholerae or E. coli enterotoxin, blocked by low concentrations of ganglioside G(M1), and destroyed by prior heating. Enterotoxin activation of adenylate cyclase was similarly affected. By contrast, prostaglandin E(1)-mediated increases in cyclic AMP were not affected by specific antitoxins or gangliosides. Combination of maximal stimulatory doses of both enterotoxins did not produce additive increases in cyclic AMP. Wash experiments suggested that both enterotoxins bind rapidly and tightly to thymocytes at 37 C. However, lowering the incubation temperature to 8 C reduced the affinity of E. coli enterotoxin but not cholera toxin for thymocytes. Results suggest that heat-labile E. coli enterotoxin and cholera enterotoxin may activate the same adenylate cyclase enzyme by similar mechanisms.     

Differential detection of cholera enterotoxin and Escherichia coli heat-labile enterotoxin by enzyme-linked immunosorbent assays with antibodies specific to the two toxins

Enzyme-linked immunosorbent assays (ELISAs) with antibodies specific to either cholera enterotoxin (CT) of Vibrio cholerae or heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli were developed to detect LT and CT, respectively. With these ELISA systems, LT and CT could be detected only with the respective specific antibody. Both antibody ELISA and ganglioside ELISA were used for differential detection of LT and CT, but the former method seemed to be more specific. By this ELISA, as little as 0.1 ng of purified LT or CT could be detected per ml. The type of toxins in fluids in intestinal loops of experimental animals challenged with living cells of either V. cholerae or LT-producing E. coli was identified correctly by this ELISA. These results suggest that the ELISA systems reported here could be used to detect and differentiate CT and LT in unknown samples; it could also be used for assaying toxins in stool specimens for the diagnosis of diarrhea due to V. cholerae or LT-producing E. coli directly, without or before bacterial isolation.     

Stimulation of Steroidogenesis in Tissue Culture by Enterotoxigenic Escherichia coli and Its Neutralization by Specific Antiserum

Y1 adrenal cells in monolayer tissue culture react to culture filtrates from enterotoxigenic strains of Escherichia coli, but not to those from nontoxigenic strains, by undergoing morphological changes similar to those inducible by cholera enterotoxin and increasing their production of steroids. Heating destroys the ability of crude preparations of the E. coli enterotoxin to effect these tissue culture changes. None of a number of culture filtrates of other enteric or enteropathogenic bacteria were capable of inducing either the morphological changes or steroidogenesis. The maximal degree of steroidogenesis achievable with E. coli enterotoxin was comparable to that of cholera enterotoxin and could not be further increased by combination of the two toxins. As with cholera enterotoxin, removal of the E. coli toxin from the tissue culture medium after an initial brief exposure of the cells to the toxin was accompanied by some decrease in maximal steroidogenesis and no changes in either the onset or the permanency of the morphological changes. Antiserum raised to one of the crude E. coli enterotoxins was capable of completely neutralizing the steroidogenic effects and preventing the morphological changes secondary to a variety of different enterotoxigenic strains of E. coli. At the concentrations tested, this antiserum was not effective in preventing these same tissue culture changes inducible by cholera enterotoxin. The results of these and other related experiments suggest that cholera and heat-labile E. coli enterotoxins have similar mechanisms of action which are dissimilar to those of the other enterotoxins thus far described and tested.     

Cytotoxic and cytotonic factors produced by Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis.

Complete toxigenicity studies were performed on 341 strains of Campylobacter spp., including 23 nonhuman isolates. Toxin profiles based on both cytotonic and cytotoxic factors were determined after analyzing responses in Vero, HeLa, CHO and Y-1 cells. Suckling mouse assays were consistently negative for all culture filtrates tested. Toxin-producing strains were frequently encountered among both the human and nonhuman strains of Campylobacter jejuni, C. coli, and C. laridis investigated. Strains isolated from outbreaks demonstrated parallels in serotype, biotype, and toxigenicity profile, although no clear association could be demonstrated. Biphasic culture conditions conducive to the production of both toxic factors were delineated for the propagation of test Campylobacter strains. Cytotonic effects of Campylobacter culture filtrates were determined in Vero and CHO cells, and cyclic AMP accumulation in cells exposed to these culture filtrates was compared with that in cells exposed to reference toxigenic strains of Vibrio cholerae and Escherichia coli. Partial neutralization of C. jejuni enterotoxin was demonstrated by using antitoxins to cholera toxin and E. coli heat-labile enterotoxin. No neutralization of C. jejuni cytotoxin could be achieved by using antitoxins to either Clostridium difficile cytotoxin or E. coli Verotoxin (0157:H7).     

Does enteropathogenic Escherichia coli produce heat-labile enterotoxin, heat-stable enterotoxins a or b, or cholera toxin A subunits?

Although most enteropathogenic Escherichia coli strains do not produce recognized enterotoxins, we wished to examine whether they produce any factors like heat-stable enterotoxin b or cholera toxin active subunits that might be missed by conventional assay methods. E. coli strains E851 (O142) and E2348 (O127) that had caused diarrhea in volunteers were negative for heat-labile enterotoxin and heat-stable enterotoxin a in Chinese hamster ovary cell and suckling mouse assays, failed to cause secretion in ligated small bowel loops from 6- to 8-week-old pigs after 4 to 5 h (used to show heat-stable enterotoxin b), and did not activate adenylate cyclase in pigeon erythrocyte lysates (used to demonstrate cholera toxin A subunit). We conclude that crude, unconcentrated culture filtrates and sonicates do not mimic heat-labile or heat-stable enterotoxins or cholera toxin or its A subunit and that enteropathogenic strains of E. coli probably have yet another mechanism or group of mechanisms by which they cause diarrhea.     

Stimulation of cyclic AMP secretion in Vero cells by enterotoxins of Escherichia coli and Vibrio cholerae.

The morphological response of Vero cells to Escherichia coli heat-labile enterotoxin was similar to that of cholera toxin and was accompanied by increases in the intracellular level of cyclic AMP. The effects of both enterotoxins were enhanced by the presence of phosphodiesterase inhibitor and inhibited by heat or specific antisera. Accumulation of cyclic AMP preceded the morphological response.     

Vero response to a cytotoxin of Escherichia coli.

A cytotoxin was found in culture filtrates of a number of Escherichia coli strains that differed from the known heat-stable and heat-labile enterotoxins of E. coli. It was cytotoxic for Vero but not for Y-1 or CHO cells, and its effect on Vero was distinctly different from that of heat-labile enterotoxin. It was labile to heat and antigenically different from heat-labile enterotoxin, and membrane filtration indicated a molecular weight of 10,000 to 30,000.     

Biological similarity of enterotoxins of Vibrio cholerae serotypes other than type 1 to cholera toxin and Escherichia coli heat-labile enterotoxin

Vibrio cholerae serotypes other than type 1, the so-called NAG vibrios, have been recognised as an important cause of diarrhoea. A few of them have been shown to produce an enterotoxin similar, immunologically and physiologically, to cholera toxin; and cholera toxin has been shown to be structurally, functionally and immunologically identical with Escherichia coli heat-labile toxin (LT). The present investigation has demonstrated biological similarities among cholera toxin, E. coli LT and enterotoxins produced by strains of V. cholerae of 59 serotypes other than 1, in the biological models, rabbit ileal loops and rabbit skin. Culture filtrates of almost all the strains were neutralised completely and all filtrates showed some neutralisation, in enterotoxic action and increase of permeability, by cholera antitoxin and E. coli LT antiserum. The partial neutralisation observed in a few strains was probably due to high concentrations of identical toxin rather than the presence of other toxic substances.     

A new heat-labile cytolethal distending toxin (CLDT) produced by Campylobacter spp

A new heat-labile toxin cytolethal to CHO, Vero, HeLa, and HEp-2 cells and negative in Y-1 cells has been demonstrated in culture filtrates of many strains of Campylobacter spp. This new toxin was termed a cytolethal distending toxin (CLDT) to reflect the progressive cell distention and eventual cytotoxicity observed in all sensitive tissue cells. CLDT was distinct from previously reported cytotoxins and cholera-like enterotoxin produced by some Campylobacter spp. Since CHO elongation induced by either the Campylobacter enterotoxin or CLDT could not be differentiated after 24 h incubation, continuation of the assay for 96 h was essential for observation of CLDT-associated progressive morphological changes and cytolethal events. Specific assay conditions were required for demonstration of CLDT in Vero, HeLa, and HEp-2 cells. A 31-fold increase in cyclic AMP levels was observed in CHO cells exposed for 24 h to CLDT of catalase negative or weak positive Campylobacter. CLDT was detected in culture filtrates from strains of Campylobacter jejuni, C. coli, C. fetus subsp. fetus, C. laridis and catalase negative or weak positive Campylobacter. Of 718 strains investigated from both human and animal isolations, 295 (41%) were found to produce this toxin. Campylobacter CLDT was negative in adult rabbit ligated ileal loops, suckling mouse and rabbit skin tests. Hemorrhagic responses were observed in rat ligated ileal loop tests of CLDT-positive cultures. The new CLDT toxin could only be neutralized by homologous rabbit antitoxin, was trypsin-sensitive, nondialyzable and over 30,000 in molecular weight. CLDT-producing strains were observed in many serogroups and biotypes of Campylobacter spp. The strains tested originated from many countries and no clear association of toxigenicity with serotype or biotype could be established.     

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.     

Heat-labile enterotoxin can be released from Escherichia coli cells by host intestinal factors.

The heat-labile enterotoxin (LT) of Escherichia coli is localized in the periplasm of the bacterial cell. Growth of an LT-producing strain of E. coli in the presence of bile salts, in concentrations within the range found in the human small intestine, caused leakage of LT into the culture medium. This leakage could be augmented by the presence in the medium of trypsin and by minimizing the concentration of free iron.     

Urea-induced release of heat-labile enterotoxin from Escherichia coli.

Urea induces the release of heat-labile enterotoxin (LT) from cells of LT-producing Escherichia coli strains. Optimal conditions were defined by using the checkerboard immunoblotting system. LT release was highest when E. coli cells were incubated in 8 M urea, pH 8.0, at 37 degrees C in a water bath for 30 min. Urea was more effective than polymyxin B in inducing the release of LT antigen from E. coli; the activity of LT from urea-treated cells was seven times that of LT from polymyxin B-treated cells. Urea also increased the antigenic and biological reactivities of purified LT. This procedure is potentially applicable for the detection of LT-producing E. coli strains in the clinical laboratory.     

Rapid detection by a coagglutination test of heat-labile enterotoxin in cell lysates from blood agar-grown Escherichia coli.

Colonies of Escherichia coli from blood agar plates were suspended and lysed in saline containing polymyxin B and a detergent (Triton X-100). The lysates were assayed for heat-labile enterotoxin (LT) by a coagglutination test (coa-test). The coa-test reagent consisted of Formalin-treated and heat-killed cells of Staphylococcus aureus, strain Cowan 1, sensitized with a high-titer rabbit anti-LT serum. Purified LT was detected in the coa-test at the nanogram level (2 to 5 ng), whereas larger amounts of cholera toxin (50 ng) were required to give a positive test. The coa-test was compared with the CHO cell test for detection of LT among E. coli strains isolated from human and animal stool cultures. The results of the coa-test and the CHO cell test correlated with 63 of 67 strains of human origin. Six of nine animal strains, defined as LT positive by the CHO cell test, gave positive results in the coa-test. We conclude that the coa-test is probably accurate and sensitive enough to be used in routine diagnosis of LT-producing E. coli strains isolated from human stool cultures. No special laboratory equipment is required, which makes the coa-test suited for diagnosis of enterotoxigenic E. coli in small hospital laboratories and in developing countries.     

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.     

Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant

Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects.     

Escherichia coli heat-labile enterotoxin: comparison of antitoxin assays and serum antitoxin levels.

The mouse adrenal tumor cells (Y-1 strain) and the Chinese hamster ovary cells, two routinely used tissue culture assays for Escherichia coli heat-labile enterotoxin (LT), were used to detect serum antitoxin responses in culture-positive patients from several well-defined sources. There was no correlation between a significant antitoxin response and isolation of LT-producing E. coli in two "domestic" diarrheal outbreaks. Serum samples from a third group of individuals in a rural cholera-endemic area consistently demonstrated significant rises in neutralizing antibody to LT.     

Detection of Escherichia coli enterotoxins in stools.

We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method.     

Interleukin-8 response in an intestinal HCT-8 cell line infected with enteroaggregative and enterotoxigenic Escherichia coli

This study examined the interleukin-8 (IL-8) response of the intestinal adenocarcinoma HCT-8 cell line to infection with enteroaggregative and enterotoxigenic Escherichia coli pathotypes isolated from patients with travelers' diarrhea. Individual diarrheagenic E. coli strains (enteroaggregative E. coli [EAEC]; n = 30), heat-stable enterotoxin (ST)-producing enterotoxigenic E. coli (ETEC ST; n = 11), heat-labile enterotoxin (LT)-producing enterotoxigenic E. coli (ETEC LT; n = 10), and ST- and LT-producing enterotoxigenic E. coli (ETEC ST:LT; n = 8) were coincubated with HCT-8 cells for 3 h. Tissue culture supernatants were assayed for IL-8 content by enzyme-linked immunosorbent assay. Fifty percent of EAEC (72% of those EAEC carrying the virulence factors aggR, aggA, and aspU and 40% of those EAEC not carrying virulence factors) and 64% of ETEC ST elicited IL-8 production. In contrast, 10% of ETEC LT elicited the production of IL-8 above baseline. These results suggest that (i) the HCT-8 cell line infection model can be used as a tool to differentiate proinflammatory E. coli from noninflammatory isolates; (ii) EAEC has a heterogeneous ability to induce the production of IL-8, and this may be associated with the presence of virulence factors; and (iii) ETEC ST can elicit an inflammatory response and helps explain our earlier findings of increased fecal IL-8 in patients with ETEC diarrhea.     

Identification of enterotoxigenic Escherichia coli in patients with diarrhea in Asia with three enterotoxin gene probes.

A total of 984 enterotoxigenic Escherichia coli (ETEC) and 733 non-ETEC isolated from patients with diarrhea in Asia (one isolate per patient) were examined for homology with radiolabeled fragments of DNA encoding heat-labile toxin (LT) or heat-stable toxin of porcine (ST-P) or human (ST-H) origin. A total of 246 ETEC that produced LT and ST as determined by the Y-1 adrenal and suckling mouse assays were homologous with the LT probe. Of these 246 LTST ETEC, 156 (63%) were homologous with the ST-H, 46 (19%) were homologous with the ST-P, and 44 (18%) were homologous with both probes. A total of 401 LT ETEC were homologous with the LT probe. Of 337 ST ETEC identified by the suckling mouse assay, 244 (72%) were homologous with the ST-H, 84 (25%) were homologous with the ST-P, and 9 (3%) were homologous with both probes. None of the 733 isolates that were non-enterotoxigenic as determined by the Y-1 adrenal and suckling mouse assays was homologous with genes coding for enterotoxin. Four isolates (not included among the 984 ETEC examined) that were initially considered to produce LT because sterile culture supernatants produced rounding of Y-1 adrenal cells were not homologous with the LT probe. The sterile culture supernatants of these four isolates caused rounding after 8 h and subsequent destruction after 24 h of Y-1 adrenal tissue cultures. This effect was not inhibited by convalescent human cholera antiserum, Swiss Serum Institute cholera antitoxin, or antiserum to purified LT. These isolates were also negative in the Biken test previously used to identify LT-producing E. coli. The DNA hybridization technique with three enterotoxin gene probes was developed as a specific technique to identify ETEC in large numbers of specimens in Asia.