Activity Of Enterotoxin Research Articles

Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.

Effect of heat-stable enterotoxins ofEscherichia coliandYersinia enterocoliticaon cyclic guanosine 3′, 5′-monophosphate levels of cultured cells

The cyclic guanosine 3′, 5′-monophosphate levels of cultured cells including CCL-6 cells and L cells were independent of heat-stable enterotoxin activity of Escherichia coli but elevated by heat-stable enterotoxin of Yersinia enterocolitica.

The effect of chemical modification on the activity of heat stable enterotoxin from Escherichia Coli

The effect of chemical modification on the activity of heat stable enterotoxin from <i>Escherichia Coli</i>

Immunological properties of porcine enterotoxigenic Escherichia Coli heat-stable (ST) enterotoxins

Cross-linking semipurified and purified heat-stable enterotoxin (2023 ST) to carrier proteins with glutaraldehyde pigs and rabbits were immunized. With ST antisera thus raised, a relatively simple, two-step method for isolation of ST is described. The method involved immunoadsorbent column chromatography procedure, and elution of the retained material with 6 M urea at pH 3.0 yielded purified ST with enterotoxin activity, controlled in suckling mouse test. ST purity was checked with a special staining technique in PAGE, anionic-exchange chromatography and immunodiffusion.

Local intestinal immune response to Escherichia Coli heat-labile (LT) enterotoxin: LT antitoxin levels in healthy, diseased and antigen-fed pigs

With purified LT toxin and IgA, specific anti-LT enterotoxin activity was demonstrated in small intestinal contents of 27 pigs. After 60 days of age, rise in intestinal LT antitoxin titer was observed. Feeding LT containing E. coli antigen increased LT antibody levels in the intestinal secretions, but decreased antibody titers in sera. In post-weaning E. coli diarrhea LT antibody levels in intestinal secretions and sera decreased significantly. This phenomenon can be related to the occurrence of the frequently observed post-weaning E. coli diarrhea.

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.

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.

Cross-neutralization of heat-stable enterotoxin activity of enterotoxigenic Escherichia coli and of Yersinia enterocolitica

Enterotoxigenic Escherichia coli produces two kinds of enterotoxin, heat-labile (LT) and heat-stable (ST). ST-like enterotoxin is reported to be produced by other bacteria, such as Yersinia enterocolitica [1 ], Klebsiella pneumoniae [2] and Enterobacter cloacae [3]. Previously, we suggested an immunological relatedness between E. coli ST and Y. enterocolitica ST by demonstrating the neutralization of crude E. coli ST by anti-Y, enterocolitica ST. As a confirmation, we demonstrate in this paper the cross-neutralization of purified ST preparations of E. coli and Y. enterocolitica. a 5-liter minijar fermentor equipped with a pHcontrol unit, at 25°C for 48 h with constant aeration (3 liters/rain) with stirring (400 rev./min).

Cross-neutralization of heat-stable enterotoxin activity of enterotoxigenic Escherichia coli and of Yersinia enterocolitica

Cross-neutralization of heat-stable enterotoxin activity of enterotoxigenic Escherichia coli and of Yersinia enterocolitica

Inhibition of protein synthesis by a tryptic polypeptide of Clostridium perfringens type a enterotoxin

The biological activity of Clostridium perfringens enterotoxin can be tested more precisely and with a much higher sensitivity by using the inhibition of protein synthesis by Vero cells, rather than the guinea pig skin test. Tryptic peptides of the enterotoxin produced in the presence of different concentrations of sodium dodecyl sulfate (0–1%) have been tested for biological activity (Vero cells) and inhibitory effect on cell-free protein synthesis (rabbit reticulocyte lysate). A fraction of tryptic peptides, about 16000 daltons, was able to inhibit the cell-free protein synthesis, while the native enterotoxin had no such effect. The 16 kDa fraction had, however, lost the ability to disrupt the Vero cells (normal biological activity). It is probable that the enterotoxin has the double function (A and B chain), known from several other toxins, confined in its single polypeptide chain.

Jejunal cAMP-activated sodium secretion via deconjugated bile salts and fatty acids

Jejunal cAMP-activated sodium secretion associated with in vivo intraluminal bile salts and free fatty acid accumulation in the small bowel was studied. Wistar rats (70–90 g) were treated with mecamylamine HCl (20 mg/kg twice daily, × 3 days); this ganglionic blocking agent allows bacterial overgrowth of the small bowel with colonic anaerobes. Controls were given 0.9% NaCl. Half of each group was fed 4% cholestyramine resin in a commercial lab chow; the other half received the same diet without this resin. The contents of the small intestine were assayed for free fatty acids, bile salts, and enterotoxin activity. Jejunal sodium and 3- O-methyl- d-glucose transport rates were assessed by in vivo perfusions. Jejunal mucosa was studied for ATPases and cAMP. Mecamylamine-treated rats had significantly higher jejunal luminal concentrations of free fatty acids and bile salts. Deconjugated bile salts were notably elevated, principally cholate. There was no enterotoxic activity detectable. Mecamylamine treatment was also associated with jejunal sodium secretion and a three-fold increase in mucosal cAMP concentrations, while control rats absorbed sodium and had no alterations in cAMP levels. Dietary cholestyramine treatment ameliorated the intestinal derangements induced by mecamylamine. Resin-treated rats had reduced luminal free fatty acids and deconjugated bile salts, while sodium was absorbed in the jejunum and mucosal cAMP levels were comparable to controls. Mecamylamine treatment did not alter (Na +-K +)-ATPase activity or jejunal transport of 3- O-methyl- d-glucose at a 4 m m concentration. These data indicate that during small bowel overgrowth with colonic bacteria following mecamylamine treatment in rats, there is a cAMP-mediated sodium secretion associated with increased luminal free fatty acids and deconjugated bile salts.

Catabolite repression of Escherichia coli heat-stable enterotoxin activity.

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.

Nad-glycohydrolase activity of staphylococcal enterotoxin a (Sea)

1. 1. Staphylococcal enterotoxin A catalyzes enzymatic hydrolysis of NAD to nicotinamide (NA) and adenosine diphosphatribose (ADP-ribose). 2. 2. The kinetic parameters of the NAD-glycohydrolase reaction catalyzed by SEA were obtained. 3. 3. The reaction characterized by K m = 2.5 × 10 −3 M and V max = 70–80 nmol mg −1 min −1. 4. 4. The graphic analysis of the kinetic parameters of the reaction showed that the hydrolysis of NAD catalyzed by SEA is a reaction of the first order.

Linkage of heat-stable enterotoxin activity and ampicillin resistance in a plasmid isolated from an Escherichia coli strain of human origin.

In an Escherichia coli strain of human origin, ampicillin resistance and heat-stable enterotoxin activity were shown by EcoRI restriction endonuclease and genetic analysis to be in an 80-megadalton plasmid.

Isolation, Enumeration, and Characterization of Aeromonas from Polluted Waters Encountered in Diving Operations

Counts of total viable, aerobic, heterotrophic bacteria, indicator organisms, and Aeromonas spp. were made at a diver training site on the Anacostia River in Washington, D.C. The numbers of Aeromonas cells in Anacostia River sediment and water increased during periods of elevated water temperature, to maxima of 4 x 10(5) cells per g of sediment and 300 cells per ml of water. Correspondingly, Aeromonas counts dropped 2 to 4 logs as the water temperature decreased to 0 to 0.5 degrees C. Cultures taken by sterile swabs from the ears and face masks of divers after a 30-min swim in the Anacostia River yielded bacterial types and numbers similar to those found in the river. The nasal passages of the divers apparently did not become contaminated by swimming, possibly because of the protective effect of the face masks used by the divers. Properties associated with virulence in Aeromonas hydrophila and Aeromonas sobria strains isolated from the river, sediment, and divers were investigated. Nearly 40% of the strains of both species collected during the study produced cytotoxic activity for mouse Y-1 adrenal cells, as well as elastase. Enterotoxin activity, as detected by the Y-1 assay, was observed in 3% (1 of 35) of the strains of A. sobria and in 6% (19 of 330) of the A. hydrophila strains. Fluid accumulation in rabbit ileal loops induced by both species of Aeromonas varied greatly among the 17 strains examined. Fluid accumulation of at least 0.4 ml/cm was correlated with positive cytotoxin- or enterotoxin-like response in the Y-1 tissue culture assay.

Passive protection of lambs against enteropathogenic Escherichia coli: role of antibodies in serum and colostrum of dams vaccinated with K99 antigen.

Lambs from suckling ewes vaccinated with the K99 antigen were resistant to challenge with K99-positive enteropathogenic Escherichia coli. Serum and colostrum from these ewes were compared with samples from control ewes to establish methods for monitoring vaccination and to determine the mechanism of protection. Vaccination stimulated production of K99 antibodies. These could be detected by an indirect haemagglutination test and a haemagglutination-inhibition test. Antiglobulin and gel-diffusion tests were less reliable. Experiments with brush-border cells from calf intestine showed that the antibodies were associated with anti-adhesive activity. The antibodies were predominantly IgG and did not neutralise the activity of heat-stable enterotoxin. It was concluded that neutralisation of the adhesive properties of the K99-positive E. coli by colostral antibodies significantly contributed to the resistance of the lambs from vaccinated ewes.

Escherichia coli heat-stable enterotoxin: rapid method of purification and some characteristics of the toxin.

In this study we used Escherichia coli strain F11(P155) of porcine origin. The heat-stable enterotoxin (ST) was produced with a batch fermentor under agitation (500 rpm) and forced aeration (5 liters/min) in Casamino Acids yeast extract medium containing 0.2% glucose. The pH varied from 7.2 to 7.8. The maximum amount of ST was obtained after 7 h of growth. ST was purified by ammonium sulfate precipitation, ultrafiltration on Amicon membranes, and chromatography in Bio-Gel P-4. The enterotoxin, which was purified approximately 1,000 times, was active at nanogram levels. On 20% polyacrylamide gel electrophoresis, ST exhibited an Rf of 0.6 ST was recovered from the gel slices by eluting in buffer and testing the activity in suckling mice, since no band appeared on the gel after staining with Coomasie brilliant blue R, Schiff reagent, or red oil. ST was resistant at pH 2 to 10 and at 100 degrees C for 15 min, but it was inactivated at 121 degrees C; it did not lose biological activity after treatment with pronase, lipase, or amylase. In suckling mice antiserum obtained from rabbits or goats immunized with ST neutralized the enterotoxin activity of a cell-free supernatant of purified ST.

Simplification of methods for the production and storage of specimens to be tested for heat-stable enterotoxin of Escherichia coli

Experiments with the infant mouse test demonstrated that there is no need of shaking for heat-stable Escherichia coli enterotoxin production when low volume of medium per volume of flask ratios are used in stationary cultures. Centrifugation and filtration of the cultures to be tested are not necessary either, and Merthiolate (1:10,000) used as preservative has no deleterious effect on heat-stable enterotoxin activity. Based upon these findings, some modifications of the procedures for production and storage of heat-stable enterotoxin preparations are suggested. Standardized pieces of filter papers are wetted with Merthiolated stationary cultures which are to be assayed for heat-stable enterotoxin activity by the infant mouse test. From dried filter papers, heat-stable enterotoxin can be eulted unaltered up to 2 months after specimen preparation. With the proposed modifications, even modestly equipped laboratories will be able to carry out the infant mouse test or at least to prepare specimens to be assayed by more specialized laboratories.

Isolation of the membranes of an enterotoxigenic strain of Escherichia coli and distribution of enterotoxin activity in different subcellular fractions

The intracellular localization of enterotoxin in Escherichia coli AP1, a strain of porcine origin which produces high levels of heat-labile, but no heat-stable enterotoxin, has been examined. The cytoplasmic and outer membranes of this strain both contained enterotoxin activity, while the membranes isolated from a serologically related non-enterotoxigenic strain ( E. coli AP2) also of porcine origin, did not show enterotoxin activity. The periplasmic fraction isolated from the enterotoxigenic strain contained considerable enterotoxin activity, but this activity was associated with outer membrane fragments present in the periplasmic fraction. Thus, of the total cellular enterotoxin activity, about 55%, 15% and 30% were present in the outer membrane, cytoplasmic membrane and the cell cytoplasm, respectively. The specific activity of enterotoxin was 20 units per mg protein in the cytoplasm and 90 and 150 units per mg protein in the cytoplasmic and outer membranes, respectively.

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.