Heat-labile enterotoxin: beyond G(m1) binding.
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ABSTRACT:
SUBMITTER: Mudrak B
PROVIDER: S-EPMC3153253 | biostudies-literature | 2010 Jun
REPOSITORIES: biostudies-literature
Publications
Enterotoxigenic Escherichia coli (ETEC) is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside G(M1), the toxin's host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified withi ...[more]
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Project description:ObjectivesEnterotoxigenic Escherichia coli (ETEC) is one of the most common agents of diarrhea among other bacterial agents. Designing and producing vaccine against these bacteria is one of the major purposes of World Health Organization (WHO). Due to presence of diverse clones of ETEC strains in the world, the use of global vaccines for ETEC infection is controversial. B subunit of heat labile toxin (LTB) was introduced as a vaccine candidate molecule by several investigators. The expression of LTB gene isolated from a local bacterial strain and investigation of its immunological property was the objective of this study.Materials and methodsLTB gene was isolated from a local isolated ETEC, cloned and expressed using pET28a expression vector. For LTB gene expression, the three main expression parameters (IPTG concentration, time and temperature of induction) were investigated. The recombinant protein was purified (>95%) with Ni-NTA column using 6XHis-tag and used as an antigen in ELISA test.ResultsThe immunological analyses showed production of high titer of specific antibody in immunized mice. Anti LTB Antibody could bind to whole toxin and neutralize the toxin through inhibition of its binding to the Ganglioside M1 receptor.ConclusionThe recombinant LTB protein is a highly immunogenic molecule. Considering the LTB role in ETEC pathogenesis, it can be taken into account as one of the most important components of vaccines against local ETEC.
| S-EPMC3279785 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli (ETEC) is a leading cause of traveler's diarrhea worldwide. One major virulence factor released by this pathogen is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. After export, LT binds to lipopolysaccharide (LPS) on the bacterial surface. Although the residues responsible for LT's binding to its host receptor are known, the portion of the toxin which mediates LPS binding has not been defined previously. Here, we describe mutations in LT that impair the binding of the toxin to the external surface of E. coli without altering holotoxin assembly. One mutation in particular, T47A, nearly abrogates surface binding without adversely affecting expression or secretion in ETEC. Interestingly, T47A is able to bind mutant E. coli expressing highly truncated forms of LPS, indicating that LT binding to wild-type LPS may be due primarily to association with an outer core sugar. Consequently, we have identified a region of LT distinct from the pocket involved in eukaryotic receptor binding that is responsible for binding to the surface of E. coli.
| S-EPMC2681808 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli is a major cause of acute diarrheal illness worldwide and is responsible for high infant and child mortality rates in developing nations. Two types of enterotoxins, one heat labile and the other heat stable, are known to cause diarrhea. The expression of soluble heat-labile toxin is subject to catabolite (glucose) activation, and three binding sites for cAMP receptor protein (CRP or CAP) were identified upstream and within the toxin promoter by DNase I footprinting. One CRP operator is centered at -31.5, thus encompassing the promoter's -35 hexamer. Potassium permanganate footprinting revealed that the occupancy of this operator prevents RNA polymerase from forming an open complex in vitro. However, the operator centered at -31.5 is not sufficient for full repression in vivo because the deletion of the other two CRP binding sites partially relieved the CRP-dependent repression of the heat-labile toxin promoter. In contrast to heat-labile toxin, CRP positively regulates the expression of heat-stable toxin. Thus, the conditions for the optimal expression of one enterotoxin limit the expression of the other. Since glucose inhibits the activity of CRP by suppressing the pathogen's synthesis of cyclic AMP (cAMP), the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. In addition, our results suggest that the host may also modulate enterotoxin expression because cells intoxicated with heat-labile toxin overproduce and release cAMP.
| S-EPMC2632052 | biostudies-literature
Project description:UnlabelledCholera toxin (CT) and the related heat-labile enterotoxins (LT) of Escherichia coli have been implicated as adjuvants in human therapies, but reactivity upon intranasal delivery dampened efforts to develop other clinical applications. However, each CT family member variant has unique biological properties that may warrant development as therapeutic platforms. In the current study, a nontoxic variant of the heat-labile enterotoxin IIa (LTIIa) was engineered to deliver heterologous, functional proteins into the cytosol of neurons. As proof of principle, the LTIIa variant delivered two cargos into neurons. LTIIa delivered β-lactamase efficiently into cells containing complex gangliosides, such as GD1b, as host receptors. LTIIa delivery of β-lactamase was sensitive to brefeldin A, an inhibitor that collapses the Golgi compartment into the endoplasmic reticulum, but not sensitive to treatment with botulinum neurotoxin D (BoNT/D), an inhibitor of synaptic vesicle cycling. LTIIa delivered a single-chain, anti-BoNT/A camelid antibody that inhibited SNAP25 cleavage during post-BoNT/A exposure of neurons. Delivery of functional, heterologous protein cargos into neurons demonstrates the potential of LTII variants as platforms to deliver therapies to inactivate toxins and microbial infections and to reverse the pathology of human neurodegenerative diseases.ImportanceThis study engineered a protein platform to deliver functional, heterologous proteins into neurons. The protein platform developed was a variant of the heat-labile enterotoxin IIa (LTIIa) which lacked the catalytic domain, yielding a nontoxic protein. As proof of principle, LTIIa variants delivered two functional proteins into neurons, β-lactamase and a camelid antibody. These studies show the utility of LTIIa variants to deliver therapies into neurons, which could be extended to inactivate toxins and microbial infections and potentially to reverse the progression of neurological diseases, such as Alzheimer's disease and Parkinson's disease.
| S-EPMC4542185 | biostudies-literature
Project description:Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage.
| S-EPMC10459231 | biostudies-literature
Project description:The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host's adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Acα3Gal or Neu5Gcα3Gal, e.g. the gangliosides GM3, GD1a and Neu5Acα3-/Neu5Gcα3--neolactotetraosylceramide and Neu5Acα3-/Neu5Gcα3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.
| S-EPMC9022906 | biostudies-literature
Project description: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.
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Project description:Various mutant forms of Escherichia coli heat-labile enterotoxin (LT) have been used as a mucosal adjuvant for vaccines, as it enhances immune responses to specific antigens including antigen-specific IgA antibodies when administrated intranasally or orally. We hypothesized that a detoxified mutant form of LT, LTS61K, could modulate dendritic cell (DC) function and alleviate allergen-induced airway inflammation. Two protocols, preventative and therapeutic, were used to evaluate the effects of LTS61K in a Dermatophagoides pteronyssinus (Der p)-sensitized and challenged murine model of asthma. LTS61K or Der p-primed bone marrow-derived dendritic cells (BMDCs) were also adoptively transferred into Der p-sensitized and challenged mice. Intranasal inoculations with LTS61K or LTS61K/Der p decreased allergen-induced airway inflammation and alleviated systemic TH2-type immune responses. Bronchoalveolar lavage fluid (BALF) and sera from LTS61K/Der p-treated mice also had higher concentrations of Der p-specific immunoglobulin (Ig) A than those of other groups. In vitro, BMDCs stimulated with Der p underwent cellular maturation and secreted proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)? In contrast, Der p-stimulated BMDCs that were pretreated with LTS61K showed decreased IL-6 and TNF? production and were less mature. Intratracheal adoptive transfer of LTS61K- or LTS61K/Der p-primed BMDCs into Der p-sensitized mice reduced inflammatory cell infiltration and TH2-type chemokines in BALF and alleviated airway inflammation in treated mice. LTS61K influenced DC maturation and decreased inflammatory cytokine production. Moreover, LTS61K/Der p induced increased Der p-specific IgA production to decrease allergic TH2 cytokine responses and alleviated airway inflammation in Der p-sensitized mice. These results suggest that the immunomodulatory effects of LTS61K may have clinical applications for allergy and asthma treatment.
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Project description:Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the alpha1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the alpha1 helix also resulted in loss of expression. However, a single amino acid deletion outside the alpha1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcription-translation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.
| S-EPMC2042161 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli (ETEC) is a major cause of illness and death but has no effective therapy. The heat-labile enterotoxin LT is a significant virulence factor produced by ETEC. The heat-labile enterotoxin-B (LT-B) subunit may enter host cells by binding to monosialotetrahexosylganglioside-a (GM1a), a monosialoganglioside found on the plasma membrane surface of animal epithelial cells. This research was conducted to develop conformationally comparable peptides to the carbohydrate epitope of GM1a for the treatment of ETEC. We used the LT-B subunit to select LT-B-binding peptides that structurally resemble GM1a. The ganglioside microarray and docking simulations were used to identify three GM1a ganglioside-binding domain (GBD) peptides based on LT-B recognition. Peptides had an inhibiting effect on the binding of LT-B to GM1a. The binding capacity, functional inhibitory activity, and in vitro effects of the GBD peptides were evaluated using HCT-8 cells, a human intestinal epithelial cell line, to evaluate the feasibility of deploying GBD peptides to combat bacterial infections. KILSYTESMAGKREMVIIT was the most efficient peptide in inhibiting cellular absorption of LT-B in cells. Our findings offer compelling evidence that GM1a GBD-like peptides might act as new therapeutics to inhibit LT-B binding to epithelial cells and avoid the subsequent physiological consequences of LT.
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