Cross-neutralization reactions of Escherichia coli and Vibrio cholerae enterotoxins as studied by rabbit skin inoculation test.

Neutralization of enterotoxins of Vibrio cholerae 569 B and Escherichia coli 10407 by antitoxins to V. cholerae 569 B, E. coli 334, 408-3 and 10407 was studies by intradermal inoculation test in the rabbit. Neutralization of V. cholerae enterotoxin by homologous as well as heterologous antisera of E. coli was observed, except that there was no neutralization of the enterotoxin by antiserum to E. coli 408-3 enterotoxin. Neutralization of E. coli enterotoxin to a varied extent by homologous as well as all heterologous antisera, including that of V. cholerae 569 B antitoxin, was also observed.     

Pregnancy interrupting effects of some bacterial toxins.

Embryotoxic properties of Shigella dysenteriae and Clostridium perfringens toxins, of E. coli endotoxin, V. cholerase and E. coli enterotoxins were compared in mice. E. coli endotoxin has embryotoxic effects at all stages of pregnancy. E. coli enterotoxin V. cholerae enterotoxin and Shigella dysenteriae toxin are most effective mainly at earlier stages of pregnancy. Clostridium perfringens toxin has no embryotoxic effect.     

Comparative study of the action of Vibrio cholerae enterotoxin, thermolabile Escherichia coli toxin and a Salmonella typhimurium filtrate on the small intestine of laboratory animals

The results of the comparative study of the effects produced by V. cholerae enterotoxin E. coli thermolabile toxin (TL-toxin) and S. typhimurum 415 filtrate on the loops of the small intestine of rabbits and C57BL/6 mice are presented. The results obtained in mice were considerably more uniform than those obtained in rabbits. For the first time the dose-effect curve for V. cholerae enterotoxin and TL-toxin, obtained on both animal species, was found to be bell-shaped. The concentration relationship for dibutyryl-cAMP proved to be similar in character. The authors suggest that these effects are based on the specific relationship between the cellular processes, controlling the transport of electrolytes and water, and the level of cAMP.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Characteristics of the B subunit of a thermolabile Escherichia coli enterotoxin produced by the A-B+ E. coli strain

The preparation and identification of B subunit of thermolabile enterotoxin produced by A-B+ gene-containing strain are described. The E. coli strain studied is shown to produce protein identical in its molecular properties and antigenic specificity to B subunit obtained from the whole thermolabile enterotoxin. Partial antigenic affinity between B subunits of thermolabile enterotoxin obtained from different sources and B subunit from cholera enterotoxin has been established in immunochemical studies. Electrophoretic and immunochemical analysis has confirmed the absence of A-subunit admixtures in B-subunit preparation obtained from /A-B+/E. coli strain.     

Determination of toxigenicity of Escherichia coli and Vibrio cholerae strains by radioimmunologic method

A solid phase variant of radioimmunoassay has been elaborated for screening toxin-producing strains of E. coli and V. cholerae grown on agar plates. The method is based on the ability of cholera-like toxins to be absorbed on nitrocellulose filters and their further identification with the use of homologous sera and [125I]-A protein from staphylococci. Sensitivity of the method reaches 20 pg. The proposed technique permits identification of intracellular enterotoxin and is aimed at a massive screening of E. coli strains, NAG-vibrios and V. cholerae strains for toxin production.     

Use of the toxin-tissue receptor reaction for detecting toxic substances of the causative agents of acute intestinal diseases

The possibility of using erythrocytic ganglioside diagnostic reagents (EGDR) for the detection of V. cholerae, E. coli and S. typhimurium enterotoxins in the passive hemagglutination (PHA) test has been shown. Museum strains and cultures isolated from patients with acute intestinal diseases were tested for the presence of enterotoxins. Cell-free extracts were studied by biological methods and by serological titration in the PHA test with the use of EGDR. The diagnostic reagent was found to interact only with those enterotoxins whose specific receptors were gangliosides GM1.     

Trial of different nutrient media for the production of an intracellular thermolabile enterotoxin by Escherichia coli strains H74-114 and 86

The conditions of cultivation, ensuring the maximum accumulation of intracellular thermolabile enterotoxin in the cultures of two E. coli strains of different origin, have been studied. Culture media manufactured in the USSR have been selected and the conditions of cultivation, necessary for obtaining intracellular thermolabile enterotoxin in preparative amounts, have been established. Under these conditions the yield of thermolabile enterotoxin in 1.4 mg per 1 liter of culture medium for strain H74-114 and 1.0 mg per 1 liter of culture medium for strain 86.     

Emission of carbonyl sulfide by Thiobacillus thioparus grown with thiocyanate in pure and mixed cultures

Abstract The biological activity of the heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was found to be predominantly associated with the periplasmic extract (about four-fold higher than the culture supernatant) of a recombinant E. coli (JM109) strain carrying the NAG-St toxin gene. Four molecular species of NAG-ST, two each from the periplasmic extract and culture supernatant of JM109, were purified. Amino acid sequence analysis of the four NAG-ST peptides isolated by HPLC revealed that they all differed from that of the mature 17-amino acid residue NAG-ST released by V. cholerae non-O1. The M _r-values of the peptides obtained from the periplasmic extract were 4331 and 2785, while those recovered from the culture supernatant were 3154 and 2785. It thus appears that V. cholerae NAG-ST is synthesized as larger molecules in the recombinant E. coli strain. The differences in sizes of the exported NAG-ST molecule could relate to difference in the enzyme cleavage system between E. coli and V. cholerea .     

Interserovar antitoxic immunity to Vibrio cholerae toxins

The immunochemical affinity of V. cholerae enterotoxins, serovars Inaba and Ogawa, has been shown in animal experiments on cross antitoxic immunity in the small intestine, the passive hemagglutination test and the toxin neutralization test. However, antitoxic interaction with both enterotoxins is characteristic only for antibodies to V. cholerae of serovar Inaba, while in animals immune to Ogawa toxin the choleragenic effect of enterotoxins produced by V. cholerae of both serovars in retained. The possible mechanisms of one-sided cross interserovar antitoxic immunity in cholera are discussed.     

Edema of the paws of white mice--test for assessing the activity of E. coli enterotoxins]

The authors studied the paw edema test in mice for detection of the E. coli (strain P-99) enterotoxins activity. This test proved to be simple, sensitive and reproducible; it permitted to determine the activity of thermostable and thermolabile enterotoxins and endotoxin; the mentioned test was particularly useful in testing various preparations of enterotoxins obtained during their extraction and purification.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Enterotoxigenicity of Salmonella strains of various origins

The study of the enterotoxigenicity of S. typhimurium with the use of the skin test on rabbits (to detect the delayed permeability factor) has revealed that these strains produce an enterotoxin similar to Escherichia coli thermolabile enterotoxin (TLE). Study of the enterotoxic activity of lysates obtained from 39 S. typhimurium strains and 5 S. dublin strains by sonication has revealed that 87% of S. typhimurium strains and all S. dublin strains produce an enterotoxin similar to E. coli TLE, as demonstrated by all tests used in this investigation, while 59% of S. typhimurium cultures and all S. dublin strains have been positive when tested for the capacity of producing the rapid permeability factor. "Hospital" strains and polyresistant cultures isolated from the environment (phagovar 20) are characterized by a higher rate of producing an enterotoxin similar to E. coli TLE, detected by the tests used in this investigation (90%), than antibiotic-sensitive strains of different origin (78%).     

Dynamics of biologically active enterotoxin release by E. coli]

Accumulation of E. coli enterotoxin in the Finkelshtein's culture medium in growing the cells in a 30-litre reactor was studied. Accumulation of active highly molecular enterotoxin occurred in the course of a 6-hour cultivation of E. coli, strain P-99 (O141: K85ab; K88ab: H4) in the fluid medium under aeration. Oxygen utilization, synthesis and release into the nutrient medium of pyruvic acid, and protein accumulation were observed. The preparation obtained was stable to the lyophylic drying, contained thermolabile and thermostable toxins and marked edema of mouse limbs. The data obtained were of significance for the industrial production of active enterotoxin preparations.     

The detection of a choleriform thermolabile enterotoxin in clinical strains of Proteus isolated in different infections

The capacity of Proteus strains, isolated from patients with purulent inflammatory, urological and enteric infections, for the production of choleriform thermolabile enterotoxin was studied by means of the enzyme immunoassay (EIA) with the use of antitoxic serum to Escherichia coli enterotoxin. Out of 125 strains, 27 (21.6%) showed the capacity for producing choleriform thermolabile enterotoxin in EIA experiments. The results thus obtained indicate that EIA techniques can be used, in principle, for detecting the capacity of Proteus for the production of choleriform thermolabile enterotoxin.     

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.     

霍乱cT-B和LPs-o双价抗原工程菌苗株的构建

作者将霍乱弧菌O抗原及毒素B亚单位基因片段,经DNA体外重组技术,得到了能表达双价抗原的工程菌株1046(pMG305)。经GM1-ELISA分析表明该菌株能够表达特异的霍乱CT-B抗原,且能分泌到胞外,通过菌体凝集,全细胞O抗原酶联分析和血凝抑制试验表明在1046(pMG305)菌体表面表达了霍乱的O抗原,它的脂多糖O抗原通过SDS-PAGE电泳分析,显示它表达了霍乱LPS的特征区带。小鼠腹腔免疫后用霍乱弧菌毒株攻击表明,有良好的保护作用,因此1046(pMG305)可望成为霍乱活疫苗的候选株。     

Potential use of serological methods in detecting cholera toxin

In the study of 50 Vibrio cholerae museum strains, 45 of them producing cholerigenic effect in suckling rabbits, cholera toxin, determined by means of the passive immune hemolysis (PIH) test, has been detected in the supernatant of the culture fluid of only two strains: V. cholerae 569 B, a well-known producer of cholera toxin, and V. cholerae (eltor) 1310, from whose population a toxigenic variant has been obtained by selection. To study the capacity of V. cholerae for producing toxin in vitro, in six cholerigenic strains, besides the supernatant of their culture fluids, also protein fractions, cell lysates and membrane fractions have been studied in the PIH test. In all these strains cholera toxin has been detected only in membrane fractions, which should be taken into consideration in the serological evaluation of the toxigenicity of V. cholerae.     

Detection of enterotoxic strains of Escherichia coli in children with diarrhea treated at the Child Health Center 1986-1988

Studies on detection of enterotoxigenic strains of E. coli were carried in 1986-1988. During this time 2324 rectal swabs from children with diarrhoea symptoms were tested. On the whole 701 E. coli were selected. The ability to produce thermolabile enterotoxin by E. coli strains was evaluated by three methods: Elek test, passive immunohemolysis and ELISA. Among selected E. coli strains enterotoxigenic strains accounted for 21.8% in 1986, 16.3% in 1987 and 28.2% in 1988.