scholarly journals Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography

2019 ◽  
Vol 20 (3) ◽  
pp. 703 ◽  
Author(s):  
Julie Heggelund ◽  
Joel Heim ◽  
Gregor Bajc ◽  
Vesna Hodnik ◽  
Gregor Anderluh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterotoxigenic Escherichia Coli ◽  
Resonance Spectroscopy ◽  
Carbohydrate Binding ◽  
Severe Diarrhea ◽  
Heat Labile ◽  
Causes Of Mortality ◽  
Site Mutagenesis ◽  
Toxin Uptake ◽  
Great Burden

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

scholarly journals Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis

2018 ◽  
Author(s):  
Julie E. Heggelund ◽  
Joel B. Heim ◽  
Gregor Bajc ◽  
Vesna Hodnik ◽  
Gregor Anderluh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Enterotoxigenic Escherichia Coli ◽  
Resonance Spectroscopy ◽  
Carbohydrate Binding ◽  
Heat Labile ◽  
Causes Of Mortality ◽  
Site Mutagenesis ◽  
Toxin Uptake ◽  
Great Burden

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galb4GlcNAcb6[Galb4GlcNAcb3]Galb4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding, enhancing avidity.

scholarly journals Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography

2019 ◽  
Author(s):  
Julie E. Heggelund ◽  
Joel B. Heim ◽  
Gregor Bajc ◽  
Vesna Hodnik ◽  
Gregor Anderluh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Enterotoxigenic Escherichia Coli ◽  
Resonance Spectroscopy ◽  
Carbohydrate Binding ◽  
Heat Labile ◽  
Causes Of Mortality ◽  
Site Mutagenesis ◽  
Toxin Uptake ◽  
Great Burden

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galbeta4GlcNAcbeta6[Galbeta4GlcNAcbeta3]Galbeta4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

scholarly journals A Tripartite Fusion, FaeG-FedF-LT192A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection

2011 ◽  
Vol 18 (10) ◽  
pp. 1593-1599 ◽  
Author(s):  
Xiaosai Ruan ◽  
Mei Liu ◽  
Thomas A. Casey ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Etec Strain ◽  
Severe Diarrhea ◽  
Heat Stable ◽  
Heat Labile ◽  
Young Pigs ◽  
Gnotobiotic Piglet

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT192) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT192A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT192A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT192A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrialE. colistrains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.

scholarly journals Electron Acceptors Induce Secretion of Enterotoxigenic Escherichia coli Heat-Labile Enterotoxin under Anaerobic Conditions through Promotion of GspD Assembly

2016 ◽  
Vol 84 (10) ◽  
pp. 2748-2757 ◽  
Author(s):  
Xi Lu ◽  
Enqing Fu ◽  
Yonghong Xie ◽  
Faguang Jin
Keyword(s):  
Escherichia Coli ◽  
Respiratory Chain ◽  
Electron Acceptors ◽  
Enterotoxigenic Escherichia Coli ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Severe Diarrhea ◽  
Heat Labile ◽  
Microaerobic Conditions

Heat-labile enterotoxin (LT), the major virulence factor of enterotoxigenicEscherichia coli(ETEC), can lead to severe diarrhea and promotes ETEC adherence to intestinal epithelial cells. Most previousin vitrostudies focused on ETEC pathogenesis were conducted under aerobic conditions, which do not reflect the real situation of ETEC infection because the intestine is anoxic. In this study, the expression and secretion of LT under anaerobic or microaerobic conditions were determined; LT was not efficiently secreted into the supernatant under anaerobic or microaerobic conditions unless terminal electron acceptors (trimethylamineN-oxide dihydrate [TMAO] or nitrate) were available. Furthermore, we found that the restoration effects of TMAO and nitrate on LT secretion could be inhibited by amytal or ΔtorCADand ΔnarGE. colistrains, indicating that LT secretion under anaerobic conditions was dependent on the integrity of the respiratory chain. At the same time, electron acceptors increase the ATP level of ETEC, but this increase was not the main reason for LT secretion. Subsequently, the relationship between the integrity of the respiratory chain and the function of the type II secretion system was determined. The GspD protein, the secretin of ETEC, was assembled under anaerobic conditions and was accompanied by LT secretion when TMAO or nitrate was added. Our data also demonstrated that TMAO and nitrate could not induce the GspD assembly and LT secretion in ΔtorCADand ΔnarGstrains, respectively. Moreover, GspD assembly under anaerobic conditions was assisted by the pilot protein YghG.

Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

1998 ◽  
Vol 61 (2) ◽  
pp. 141-145 ◽  
Author(s):  
HAU-YANG TSEN ◽  
LIANG-ZHAO JIAN ◽  
WAN-RONG CHI
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Skim Milk ◽  
Pcr Primers ◽  
Enterotoxigenic Escherichia Coli ◽  
Farm Animals ◽  
Target Cells ◽  
Heat Stable ◽  
Heat Labile

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT I- and ST II-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 100 cells per ml of the sample could be detected. Without the preculture step, 104 CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR System can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

scholarly journals Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

2016 ◽  
Vol 473 (21) ◽  
pp. 3923-3936 ◽  
Author(s):  
Dani Zalem ◽  
João P. Ribeiro ◽  
Annabelle Varrot ◽  
Michael Lebens ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Cholera Toxin ◽  
Carbohydrate Binding ◽  
Type I ◽  
Type Ii ◽  
E Coli ◽  
B Subunit ◽  
Heat Labile ◽  
B Subunits

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

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