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.     

Induction of Escherichia coli and Vibrio cholerae enterotoxins by an inhibitor of protein synthesis.

Enterotoxigenic Escherichia coli or Vibrio cholerae 569B (Inaba) grown in the presence of the antibiotic lincomycin, an inhibitor of protein synthesis, produced elevated levels of heat-labile enterotoxin or choleragen, respectively, as assayed by both vascular permeability factor and capacity to elicit fluid accumulation in rabbit ileal loops. This induction of enterotoxin did not reflect either a coupling of lincomycin resistance with increased enterotoxigenicity or an effect of lincomycin on cellular release of enterotoxin, since spontaneously isolated lincomycin-resistant mutants of both E. coli and V. cholerae still required lincomycin for induction, and large increases in E. coli permeability factor activity were found intracellularly as well as extracellularly. After the period of exponential growth, E. coli became refractory to induction by lincomycin, although most of the induced enterotoxin activity appeared only after this period. No increase in copy number of the enterotoxin plasmid in E. coli 711 (P307) was found in induced cells by analysis of deoxyribonucleic acid reassociation kinetics. These and other data suggest that synthesis of enterotoxin, or at least its accumulation, is normally limited by cellular factors whose synthesis is preferentially inhibited by lincomycin. A possible connection between this phenomenon and lincomycin-associated diarrhea is considered.     

Berberine inhibits intestinal secretory response of Vibrio cholerae and Escherichia coli enterotoxins.

Eight strains of laboratory mice were susceptible to subclinical infections with Cryptosporidium sp. at 1 to 4 days of age, but only a transient infection could be established at 21 days of age or older. Immunosuppression of 21-day-old mice failed to render them more susceptible to infection. Laboratory storage conditions for Cryptosporidium sp. were investigated by titration in 1- to 4-day-old mice. Storage by freezing with a variety of cryoprotectants was unsuccessful, but storage at 4 degrees C in phosphate-buffered saline or 2.5% potassium dichromate was possible for 4 to 6 months.     

Demonstration of shared and unique immunological determinants in enterotoxins from Vibrio cholerae and Escherichia coli.

Immunodiffusion and biological neutralization studies demonstrated that the heat-labile enterotoxin (LT) from Escherichia coli has antigenic determinants in common with each of the isolated subunits (A and B) of the enterotoxin (choleragen) from Vibrio cholerae. Each of the enterotoxins also possesses unique antigenic specificities. Monospecific antiserum to LT was prepared by immunization with antigens derived by immune precipitation of E. coli cell-free supernatant with isolated specific anticholeragenoid antibodies. This antiserum neutralized the biological acitivity of both LT and cholera enterotoxin and recognized antigens of both in immunodiffusion. This antiserum was adsorbed with choleragenoid to remove antibodies directed against the shared "B" immunological determinants. The neutralizing effect of the antiserum on cholera toxin was completely removed, but the neutralizing activity against the E. coli preparations was retained, although somewhat reduced. Antisera to the isolated subunits (A and B) of cholera enterotoxin neutralized the biological activity of cholera enterotoxin and LT. These antisera also recognized the homologous and heterologous antigens in immunodiffusion. Multiple forms or conformations of LT and its components may explain the diversity of the properties which have been reported for it.     

Rapid visual detection of Escherichia coli and Vibrio cholerae Heat-labile enterotoxins by nitrocellulose enzyme-linked immunosorbent assay

A modification of the enzyme-linked immunosorbent assay for a sensitive and rapid visual detection of heat-labile enterotoxins from Escherichia coli and Vibrio cholerae is described. Small amounts of bacterial supernatant fluids are bound to nitrocellulose filters which are used as sorbents in the nitrocellulose enzyme-linked immunosorbent assay. The test is based on the immunological similarity between V. cholerae and E. coli heat-labile enterotoxins. Six isolates of V. cholerae and 48 isolates of E. coli were examined for heat-labile enterotoxins by the nitrocellulose enzyme-linked immunosorbent assay and the Vero cell bioassay. With some strains, the nitrocellulose enzyme-linked immunosorbent assay was found to be more sensitive for detection of E. coli heat-labile enterotoxin than the Vero cell test. A similar result was obtained by endpoint titration of heat-labile enterotoxin-positive E. coli H10407 culture fluid in both assays. The sensitivity of the nitrocellulose enzyme-linked immunosorbent assay for the detection of purified cholera toxin was at a total level of 1 ng, which is a good result when compared with other serological assays.     

Effect of lodoxamide on the secretory response induced by Escherichia coli and Vibrio cholerae enterotoxins in infant mice.

The effect of lodoxamide tromethamine, a calcium antagonist, on intestinal fluid accumulation induced by Escherichia coli heat-stable (ST) and Vibrio cholerae (CT) enterotoxins in infant mice was investigated. The simultaneous administration of lodoxamide with ST or CT enterotoxin resulted in a significant (P less than 0.01) inhibition of the intestinal fluid response. A minimum concentration of 10(-7) or 10(-8)M lodoxamide caused an inhibition (P less than 0.01) of the ST- or CT-mediated fluid response, respectively. Treatment of infant mice with buffer or drug alone did not result in fluid accumulation. A significant inhibition of ST and CT enterotoxic activities was also observed when lodoxamide was administered 30 min before (P less than 0.02) or 30 min after (P less than 0.01) toxin challenge. These data suggest that calcium may be important in the ST- or CT-mediated induction of fluid accumulation. Further studies on the potential use of lodoxamide tromethamine in both the prophylaxis and treatment of diarrheal disease appear warranted.     

The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.

This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.     

Isolation of special antibodies which react only with homologous enterotoxins from Vibrio cholerae and Enterotoxigenic Escherichia coli.

Cholera enterotoxin (CT) and heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli share a common antigenic determinant. In addition, CT and LT each have a unique antigenic determinant. Antisera were prepared by immunoaffinity chromatography against these unique antigenic determinants, that is, antiserum that reacted with CT but not with LT and antiserum that reacted with LT but not with CT. Antiserum against the common antigenic determinant to CT and LT was also prepared by immunoaffinity chromatography. The specificities of these antisera were demonstrated by Ouchterlony double-gel diffusion tests and by neutralization of the activities of CT and LT to cause morphological changes in Chinese hamster ovary cells.     

Isolation of a factor causing morphological changes of chinese hamster ovary cells from the culture filtrate of Vibrio parahaemolyticus.

A factor changing Chinese hamster ovary cells from an oval to a spindle shape was isolated from the culture filtrate of Vibrio parahaemolyticus. It was partially purified by successive column chromatographies on diethylaminoethylcellulose, diethylaminoethyl-Sephadex A-25, hydroxylapatite, and Sephadex G-200. This factor was separated from thermostabl direct hemolysin and was heat labile.     

Assay of Escherichia coli enterotoxins by in vivo perfusion in the rat jejunum.

Assay of Escherichia coli enterotoxins by in vitro perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100 degrees C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.     

Evaluation of BW942C, a novel antidiarrheal agent, against enterotoxins of Escherichia coli and Vibrio cholerae.

BW942C, an enkephalin-like pentapeptide with anti-diarrheal activity, was tested against crude toxins of Escherichia coli and Vibrio cholerae in the Y-1 adrenal cell assay, rabbit ileal loop assay, and suckling mouse assay. The effects of BW942C on in vitro ion transport were measured in rabbit ileum mounted in Ussing chambers. In vitro, BW942C decreased basal short-circuit current (2.26 and 3.15 mueq cm-2 h-1 in experimental samples and controls, respectively; n = 7, P less than 0.05) and increased basal net Cl absorption (1.59 and 0.50 mueq cm-2 h-1 in experimental samples and controls, respectively; P less than 0.025). Net Na absorption was also increased, but not significantly. BW942C did not block the secretory response to a maximal dose of purified heat-stable toxin. BW942C directly enhanced intestinal fluid absorption. In the Y-1 adrenal cell assay, 5 mg of BW942C per ml inhibited the cytopathic effect caused by cholera toxin or heat-labile enterotoxin of E. coli. In the rabbit ileal loop assay, E. coli heat-stable toxin, E. coli heat-labile enterotoxin, and cholera toxin were inhibited 35 to 70% by administration of BW942C. With the suckling mouse model, the fluid accumulation caused by E. coli heat-stable toxin was ablated by prior treatment with BW942C. The drug is currently being evaluated in patients with acute secretory diarrhea to determine its effect on clinical symptoms.     

Adaptive response of Vibrio cholerae and Escherichia coli to nitrofurantoin.

Pretreatment with sublethal doses of nitrofurantoin induced adaptive response in both Vibrio cholerae and Escherichia coli cells as indicated by their greater resistance to the subsequent challenging doses of the same drug. Adaptive response was maximum corresponding to pretreatment drug concentrations of 0.40 microgram/ml and 0.015 microgram/ml respectively for V. cholerae OGAWA 154 (wild type) and E. coli K-12 AB 2463 (recA-) cells. Adaptive response was inhibited by chloramphenicol (100 micrograms/ml) indicating the need of concomitant protein synthesis. Induction of adaptive response in recA deficient E. coli cells indicated that it was different from the conventional "SOS" response. Melting temperature of DNA of V. cholerae cells subjected to adaptive (0.4 microgram/ml for 1 hr) and challenging (120 micrograms/ml for 1 hr) doses of nitrofurantoin (76 degrees C) was closer to that of native DNA (75 degrees C) vis-a-vis DNA isolated from nonadapted and drug treated cells (77.5 degrees C). Also, DNA isolated from V. cholerae cells subjected to adaptive and challenging doses of the drug revealed the presence of fewer interstrand cross-links (16% reversible DNA) vis-a-vis DNA from nonadapted but drug treated cells (55% reversible DNA). Photomicrographic studies revealed that V. cholerae cells that were nonadapted but drug treated grew into long filamentous forms (4.25 +/- 2.97 micron) whereas those subjected to both adaptive and challenge doses of the drug exhibited much less filamentation (2.08 +/- 0.84 micron) vis-a-vis native cells (1.42 +/- 0.5 micron). Similar results on DNA melting temperature, cross-links in DNA, and filamentation of cells were obtained for E. coli AB 2463 (recA-) cells subjected to adaptive and challenging treatments with nitrofurantoin. Almost equal degree of resistance against nitrofurantoin could be induced in both V. cholerae OGAWA 154 (wild type) and E. coli strain PJ3 (AB 1157 ada-) when these cells were pretreated with nontoxic doses of hydrogen peroxide or nitrofurantoin. Evidence obtained in this work on the nature of the nitrofuratoin induced adaptive response with particular references to the oxidative and/or alkylating DNA damages were discussed. Nitrofuratoin induced adaptive response appeared similar to that elicited by furazolidone in V. cholerae cells and appeared to be directed towards oxidative and not alkylating adaptive repair pathway.     

Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea.

We developed an adult rabbit model for enteric infection by Vibrio cholerae and enterotoxigenic Escherichia coli. The cecum of each animal was first ligated to prevent it from retaining fluid secreted by the small intestine. A temporary reversible obstruction (a slip knot tie) of the small bowel was introduced at the time of challenge and removed 2 h later. With this modification, we were able to elicit a massive and usually fatal cholera-like diarrhea in adult (3.5- to 6-lb [1.6- to 2.7-kb]) animals challenged with V. cholerae. Animals challenged with enterotoxigenic E. coli also developed diarrhea which was severe and watery but less explosive and less rapidly fatal than that produced by V. cholerae. The susceptibility of animals in this model to infection by V. cholerae was similar to the susceptibility of infant rabbits challenged intraintestinally. The death rate was almost 25% when 10(3) Vibrio cells were given and 90% or more when the dose was greater than or equal to 10(6) cells per animal. We designated this procedure the RITARD (for removable intestinal tie-adult rabbit diarrhea) model.     

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.     

Expression of the Escherichia coli lamB gene in Vibrio cholerae

A phage lambda mediated transduction system was devised to facilitate molecular analysis of Vibrio cholerae. A lamB expression plasmid, pAMH62 was introduced into Vibrio cholerae by conjugation. The resulting V. cholerae derivatives harboring pAMH62 produced substantial amounts of the LamB protein. This protein was properly inserted into the outer membrane, as suggested by (i) its localization into the cell envelope, (ii) its association with the peptidoglycan layer of the cell wall, and (iii) its function as receptor for phage lambda. In vivo packaged cosmids were efficiently transduced into these strains of V. cholerae.     

Effect of enteropathogenic Escherichia coli on adherent properties of Chinese hamster ovary cells.

Enteropathogenic Escherichia coli (EPEC) O111:H2, O119:H6, or O142:H6 caused rapid detachment of Chinese hamster ovary (CHO) cell monolayers within 2 to 4 h of cocultivation. CHO cell detachment was not promoted by nonenteropathogenic E. coli (O125:H4, O126:H27, O157:H7, and O26:H11) and could not be attributed to EPEC production of enterohemolysin or Shiga-like toxins. In contrast, EPEC strains did not promote rapid detachment of Lec1, Lec2, or Lec8 CHO cell monolayers. These CHO cell Lec mutants all express abbreviated glycan sequences on membrane glycoproteins and glycolipids. Although EPEC strains failed to alter the adherent properties of Lec2 cells lacking only terminal sialic acid groups, EPEC adherence to the Lec2 mutant was indistinguishable from that observed with wild-type CHO cells. There was also no significant difference in EPEC-induced actin accumulation or invasion of Lec2 cells. In contrast, EPEC localized adherence to Lec1 and Lec8 mutants, lacking sialyllactosamine (Lec1) or sialic acid and galactose (Lec8) sequences, was reduced by 84 and 93%, respectively. Our results suggest that lactosamine sequences [beta Gal(1-4 or 1-3)beta GlcNAc] not containing sialic acid are sufficient for EPEC adherence, actin accumulation, and invasion of CHO cells. Sialic acid groups, however, may be necessary for EPEC-mediated CHO cell detachment.     

Inhibition of enterotoxin from Escherichia coli and Vibrio cholerae by gangliosides from human milk.

Inhibitory activity of enterotoxin from Escherichia coli and Vibrio cholerae was associated with the ganglioside fraction of human milk. Both the milk fat and skim milk contained gangliosides that inhibited the toxins. The most purified milk fraction contained three glycolipid components, of which two migrated close to ganglioside GM1 on thin-layer chromatography plates. A component with a slightly different mobility from GM1 appeared to be associated with the inhibitory activity. Milk ganglioside fraction, derived from 2 ml of human milk, contained 1 to 4 micrograms of lipid-bound sialic acid and completely inhibited 0.1 micrograms of cholera toxin in rabbit intestinal loop experiments. It is suggested that human milk gangliosides, although present only in trace amounts, may be important in protecting infants against enterotoxin-induced diarrhea.     

Transfer of genes to chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNA to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk^– cells were transformed with the cloned gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk^– cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 × 10^–5 were obtained. CHO HSV- tk⁺ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10^–4 with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk^– cells could be transformed at a frequency of 10^–7 with cellular DNA isolated from CHO tk⁺ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.A preliminary account of this work has been presented at the twenty-first annual meeting of the American Society for Cell Biology, 1981 (Ref. 41).     

Functional hemizygosity for theMDH2 locus in chinese hamster ovary cells

Isolation of electrophoretic mobility shift mutants for a large number of enzyme loci in CHO cells has allowed the identification of many genes which are functionally hemizygous. To gain further insight into the nature of hemizygosity in CHO cells and the mechanisms by which it has arisen, we are attempting to determine whether hemizygous gene loci are clustered in a few localized chromosomal regions in CHO or are more generally distributed throughout the genome. Isozyme analysis of a series of CHO electrophoretic mobility shift mutants for MDH2 (malate dehydrogenase 2, EC 1.1.1.37) revealed that this locus is functionally hemizygous in CHO cells, but the locus could not be mapped by conventional approaches because of the similar electrophoretic mobilities of Chinese hamster and mouse MDH2 isozymes. Construction of intraspecific CHO x CHO hybrids using electrophoretic mobility shift mutants with secondary, selectable drug-resistance markers allowed us to determine that MDH2 is not closely linked to any previously mapped hemizygous marker loci in CHO, but is linked to allelesfor two dizygous gene loci, PGM3 and APRT, on CHO chromosome Z7. A possible genetic basis for hemizygosity of the MDH2 locus in CHO cells is discussed.