Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli

Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development.     

Purification and characterization of heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

Abstract The heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli was purified to homogeneity and its molecular and antigenic properties were compared with those of purified LTs from porcine and human enterotoxigenic Escherichia coli (LTp, LTh). The A subunit of LTc was identical to that of LTp and the B subunit of LTc was identical to that of LTh but not that of LTp, in mobility on SDS-polyacrylamide gel electrophoresis. Ouchterlony tests demonstrated that LTc is antigenically identical to LTh but not with LTp. The p I point and amino acid composition of LTc were also compared and the results suggest that chicken enterotoxigenic E. coli produced an LT similar to LTh.     

Isolation and characterization of enterotoxigenic Escherichia coli mutants that produce abnormal heat-labile enterotoxins

Abstract Bacteroides fragilis populations were separated according to the size of surface structure. Subculture of the separated populations produced cultures enriched for 3 different structures; a large capsule, a small capsule and an electron-dense layer (EDL). The ability of these subpopulations to haemagglutinate (HA) erythrocytes from a number of species was examined. Populations which produced either a large or s small capsule did not have HA activity, whereas those with an extracellular EDL did. By mixing populations with EDL and those with either the large or small capsule, the degree of HA could be altered. HA was dependent on the proportion of EDL-bearing bacteria present. Fimbriae were not observed on electron microscopy.     

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.     

Cistrons encoding Escherichia coli heat-labile toxin.

The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.     

Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli

Abstract We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the p I points of B subunits of the LThs were identical to each other, the p I points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli .     

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.     

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3',5'-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.     

Hemagglutinating activity of the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli

Abstract The hemagglutinating activity of the heat-labile enterotoxin (LT_p) isolated from porcine enterotoxigenic Escherichia coli was studied by hemagglutination inhibition. The hemagglutinating activity of LT_p was enhanced 64–512-fold with pronase- and neuraminidase-treated human erythrocytes although both intact human and sheep erythrocytes were not agglutinated by LT_p at the highest concentration used. No enhancement was found in hemagglutination of neuraminidase-treated sheep erythrocytes by LT_p. Hemagglutination of pronase-treated human type A erythrocytes induced by LT_p was inhibited by melibiose and galactose among mono-, di-, and polysaccharides used as inhibitors. Galactose was a slightly better inhibitor than melibiose. These findings suggest that LT_p is a bacterial lectin specific for galactose.     

Effect of chlorine injury on heat-labile enterotoxin production in enterotoxigenic Escherichia coli

Heat-labile enterotoxin (LT) production was examined in chlorine-injured and noninjured populations of enterotoxigenic Escherichia coli (ETEC) by passive immune hemolysis and Y-1 mouse adrenal tumor cell assays. Sublethally injured populations showed reduced LT production after 1, 2.5, and 4 h incubation in trypticase soy broth plus 0.25% glucose, pH 8.0. Reduction was observed during injury, resuscitation, and for at least 1.5 h following repair. LT levels comparable with that present in noninjured cells were found after 24 h incubation in the same medium, indicating delayed toxigenesis rather than permanent damage. Chlorinated populations failed to incorporate [14C]glucose until repair was completed suggesting a possible explanation for delayed toxin production. The results indicate a temporary loss of virulence among sublethally injured ETEC in chlorinated waters.     

Binding specificities of heat-labile enterotoxins isolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides

The binding specificities of heat-labile enterotoxins (LTp and LTh) isolated from porcine and human enterotoxigenic Escherichia coli on human erythrocytes were studied by competitive binding assays using different gangliosides as inhibitors. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1. Ganglioside GM1 was 11 and 105 times more potent than gangliosides GD1b and GM2, respectively. Gangliosides GD1a, GT1b, and GM3 were much less potent. Similar results were also obtained in competitive binding assays with the 125I-labeled B subunit of LTh and neuraminidase-treated human type B erythrocytes, and in those with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. The binding of 3H-labeled ganglioside GM1 to LTp was not effectively inhibited by galactose-beta(1----3)N-acetyl-D-galactosamine at the highest concentration used. These findings suggest that the combining sites of LTp and LTh may be specific for at least the galactose-N-acetyl-D-galactosamine-galactose (N-acetyl-neuraminic acid) portion of ganglioside GM1.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.