| 1. |
- Larena, Maximilian, et al.
(author)
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Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling
- 2015
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 194:8, s. 3829-39
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Journal article (peer-reviewed)abstract
- We have examined the molecular pathways involved in the adjuvant action of cholera toxin (CT) and two novel nontoxic molecules, multiple-mutated CT (mmCT) and double-mutant heat-labile toxin (dmLT) on human T cell responses. Human PBMCs or isolated monocytes were stimulated in vitro with CT, mmCT, or dmLT plus a polyclonal stimulus (staphylococcal enterotoxin B) or specific bacterial Ags, and effects on expression of cytokines and signaling molecules were determined. CT, mmCT, and dmLT strongly enhanced IL-17A and to a lesser extent IL-13 responses, but had little effect on IFN-gamma production or cell proliferation. Intracellular cytokine staining revealed that the enhanced IL-17A production was largely confined to CD4(+) T cells and coculture experiments showed that the IL-17A promotion was effectively induced by adjuvant-treated monocytes. Relative to CT, mmCT and dmLT induced at least 100-fold lower levels of cAMP, yet this cAMP was enough and essential for the promotion of Th17 responses. Thus, inhibition of cAMP-dependent protein kinase A was abolished, and stimulation with a cAMP analog mimicked the adjuvant effect. Furthermore, CT, mmCT, and dmLT induced IL-1beta production and caspase-1 activation in monocytes, which was associated with increased expression of key proinflammatory and inflammasome-related genes, including NLRP1, NLRP3, and NLRC4. Inflammasome inhibition with a specific caspase-1 inhibitor, or blocking of IL-1 signaling by IL-1 receptor antagonist, abrogated the Th17-promoting effect. We conclude that CT, mmCT, and dmLT promote human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent IL-1 signaling.
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| 2. |
- Lebens, Michael, 1956, et al.
(author)
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Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae
- 2016
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In: Vaccine. - : Elsevier Ltd. - 0264-410X .- 1873-2518. ; 34:18, s. 2121-2128
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Journal article (peer-reviewed)abstract
- There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage ("nicking") required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines
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| 3. |
- Rattik, Sara, et al.
(author)
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B cells treated with CTB-p210 acquire a regulatory phenotype in vitro and reduce atherosclerosis in apolipoprotein E deficient mice
- 2018
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In: Vascular Pharmacology. - : Elsevier BV. - 1537-1891. ; 111, s. 54-61
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Journal article (peer-reviewed)abstract
- Objective: Intranasal immunization with a fusion protein of the ApoB100-derived peptide p210 and the cholera toxin B subunit (CTB-p210) has previously been shown to induce mucosal tolerance and reduce atherosclerosis development, but the exact mode of action remains to be elucidated. Recent studies have indicated an important role for B cells in mucosal tolerance, in particular by induction of regulatory B (Bregs) and T cells (Tregs). In this study, we aimed to investigate if transfer of B cells pulsed with CTB-p210 can protect against atherosclerosis. Method and results: First, we studied if CTB-p210 can induce Bregs and Tregs in vitro. After pulsing B cells from Apob(tm2gy)ldlr(-/-) or Apoe(-/-) mice with CTB-p210 for 1 h and co-culturing them with naive T cells for 48 h, we observed increased expression of membrane bound TGF beta/latency-associated peptide (mTGF beta/LAP) on B cells and an increased proportion of CD25(hi)FoxP3(+) Tregs. Adoptive transfer of B cells pulsed with CTB-p210 into high-fat diet-fed Apoe(-/-) mice at 8, 10 and 12 weeks of age, reduced the plaque area in the aorta at 20 weeks of age as compared with control-treated (CTB-pOVA treated B cells or PBS) mice. Moreover, mice receiving p210-CTB treated B cells had increased levels of anti-p210 IgG antibodies. Conclusion: Our observations suggest that CTB-p210 pulsed B cells acquire a regulatory phenotype and induce Tregs in vitro. Adoptive transfer of CTB-p210, but not control-treated, B cells into Apoe(-/-) mice decreased atherosclerosis development.
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| 4. |
- Sun, Jiabin, et al.
(author)
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Deficiency in calcium-binding protein S100A4 impairs the adjuvant action of cholera toxin
- 2017
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 8:SEP
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Journal article (peer-reviewed)abstract
- The calcium-binding protein S100A4 has been described to promote pathological inflammation in experimental autoimmune and inflammatory disorders and in allergy and to contribute to antigen presentation and antibody response after parenteral immunization with an alum-adjuvanted antigen. In this study, we extend these findings by demonstrating that mice lacking S100A4 have a defective humoral and cellular immune response to mucosal (sublingual) immunization with a model protein antigen [ovalbumin (OVA)] given together with the strong mucosal adjuvant cholera toxin (CT), and that this impairment is due to defective adjuvant-stimulated antigen presentation by antigen-presenting cells. In comparison to wild-type (WT) mice, mice genetically lacking S100A4 had reduced humoral and cellular immune responses after immunization with OVA plus CT, including a complete lack of detectable germinal center reaction. Further, when stimulated in vitro with OVA plus CT, S100A4-/- dendritic cells (DCs) showed impaired responses in several CT-stimulated immune regulatory molecules including the co-stimulatory molecule CD86, inflammasome-associated caspase-1 and IL-1ß. Coculture of OVA-specific OT-II T cells with S100A4-/- DCs that had been pulse incubated with OVA plus CT resulted in impaired OT-II T cell proliferation and reduced production of Th1, Th2, and Th17 cytokines compared to similar cocultures with WT DCs. In accordance with these findings, transfection of WT DCs with S100A4-targeting small interfering RNA (siRNA) but not mock-siRNA resulted in significant reductions in the expression of caspase-1 and IL-1ß as well as CD86 in response to CT. Importantly, also engraftment of WT DCs into S100A4-/- mice effectively restored the immune response to immunization in the recipients. In conclusion, our results demonstrate that deficiency in S100A4 has a strong impact on the development of both humoral and cellular immunity after mucosal immunization using CT as adjuvant. © 2017 Sun, Holmgren, Larena, Terrinoni, Fang, Bresnick and Xiang.
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| 5. |
- Terrinoni, Manuela, et al.
(author)
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A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae
- 2018
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In: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 84:4
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Journal article (peer-reviewed)abstract
- Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro) lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30 degrees C but not at 37 degrees C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39 degrees C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli. The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation. IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.
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| 6. |
- Terrinoni, Manuela, et al.
(author)
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A thermostable, dry formulation inactivated Hikojima whole cell/cholera toxin B subunit oral cholera vaccine
- 2023
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In: Vaccine. - 0264-410X. ; 41:21, s. 3347-3357
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Journal article (peer-reviewed)abstract
- The feared diarrheal disease cholera remains an important global health problem. Use of oral cholera vac-cine (OCV) from a global stockpile against both epidemic and endemic cholera is a cornerstone in the World Health Organisations (WHOs) global program for "Ending cholera by 2030". Three liquid inacti-vated whole-cell OCVs (Dukoral (R), ShancholTM, and Euvichol-Plus (R)) are WHO prequalified and have proved to be safe and effective. However, their multicomponent composition and cold-chain requirement increase manufacturing, storage and transport costs. ShancholTM and Euvichol-Plus (R) OCVs used in WHOs global vaccine stockpile also lack the protective cholera toxin B-subunit (CTB) antigen present in Dukoral (R), which results in suboptimal efficacy.WHOs Global Task Force on Cholera Control (GTFCC) has identified a thermostable, dry formulation vaccine as a priority for further OCV development. We describe here the development of such a vaccine, based on a lyophilized mixture of a single strain of formalin-killed Hikojima bacteria together with a low-cost, recombinantly produced CTB. The new vaccine, which is easy and inexpensive to manufacture, could be stored for at least 26 months at 25 degrees C and for at least 8 months at 40 degrees C with preservation of cell mor-phology and with no loss of protective Ogawa and Inaba lipopolysaccharides or CTB. It also proved to be well tolerated and to have equivalent oral immunogenicity in mice as ShancholTM and Dukoral (R) OCVs with regard to both serum and intestinal-mucosal antibody responses.
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| 7. |
- Terrinoni, Manuela
(author)
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Novel approaches to mucosal vaccine development Strategies in vaccine antigen production, construction of a novel mucosal adjuvant and studies of its mode of action
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- Although most infections begin at a mucosal surface and may be prevented by effective vaccine stimulation of the local mucosal immune system, there are so far only a few mucosal vaccines available for human use. This thesis spans several areas that are important for future development of mucosal vaccines. Future vaccine development will depend in part on the efficient production of recombinant antigens produced in bacterial expression systems. To avoid current problems with the use of antibiotics to maintain expression plasmids, an E. coli strain capable of producing recombinant proteins using vectors maintained without the need antibiotic was generated. The method is based on deletion of the essential lgt gene encoding a (pro)lipoprotein glyceryl transferase and complementing it with an expression vector carrying the non-homologous lgt gene from V. cholerae. A similar V. cholerae lgt-deleted strain was also constructed using the E. coli lgt gene for complementation. The strains had similar growth and production characteristics as their wild-type counterparts but maintained their expression plasmids without the need for antibiotics. The system was used to express two recombinant vaccine proteins, cholera toxin B subunit and a fusion protein for vaccination against atherosclerosis. In the development of mucosal vaccines, it is often important to enhance immune responses using adjuvants, since most mucosally administered antigens are poorly immunogenic. Cholera toxin (CT) is the most powerful mucosal adjuvant known but is too toxic for human use. A mutated CT derivative (mmCT) was constructed and expressed in an engineered strain of V. cholerae. mmCT induced 1000 times less cAMP than native CT in a mouse thymocyte toxicity assay, was non-toxic in an infant mouse model and yet retained similar adjuvant properties as native CT. We suggest that mmCT is a promising candidate for use in future mucosal vaccines. The mode of adjuvant action of mmCT and native CT was investigated using human and mouse antigen-presenting cells, which are primary target cells for adjuvants. Both molecules were found to activate cyclic AMP/protein kinase A-dependent canonical NF-kB signaling associated with inflammasome activation. The activation of these pathways was found to induce expression of two immunomodulatory proteins, THSB1 and ITGB1, as well as increased expression and activation of IL-1β, a cytokine which has been shown to play an important role for the adjuvant action of CT and mmCT.
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| 8. |
- Terrinoni, Manuela, et al.
(author)
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Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-beta 1 as strongly upregulated molecules involved in adjuvant activity
- 2019
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Journal article (peer-reviewed)abstract
- Cholera Toxin (CT) as well as its related non-toxic mmCT and dmLT mutant proteins have been shown to be potent adjuvants for mucosally administered vaccines. Their adjuvant activity involves activation of cAMP/protein kinase A (PKA) signaling and inflammasome/IL-1 beta pathways in antigen presenting cells (APC). To get a further understanding of the signal transduction and downstream pathways activated in APCs by this group of adjuvants we have, employing quantitative proteomic analytic tools, investigated human monocytes at various time points after treatment with CT. We report the activation of three main biological pathways among upregulated proteins, peaking at 16 hours of CT treatment: cellular organization, metabolism, and immune response. Specifically, in the further analyzed immune response pathway we note a strong upregulation of thrombospondin 1 (THBS1) and integrin beta 1 (ITGB1) in response to CT as well as to mmCT and dmLT, mediated via cAMP/PKA and NFKB signaling. Importantly, inhibition in vitro of THSB1 and ITGB1 in monocytes or primary dendritic cells using siRNA abrogated the ability of the treated APCs to promote an adjuvant-stimulated Th17 cell response when co-cultured with peripheral blood lymphocytes indicating the involvement of these molecules in the adjuvant action on APCs by CT, mmCT and dmLT.
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| 9. |
- Terrinoni, Manuela, et al.
(author)
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Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NF kappa B Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT
- 2019
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
-
Journal article (peer-reviewed)abstract
- Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NF kappa B, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NF kappa B-/- mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1 beta, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NF kappa B activation reporter system, we show that CT induced NF kappa B signaling in human monocytes, and that inhibition of the cyclic AMP-protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NF kappa B. In a human monocyte-CD4(+) T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NF kappa B signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NF kappa B pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NF kappa B signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.
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| 10. |
- Terrinoni, Manuela, et al.
(author)
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Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT
- 2019
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
-
Journal article (peer-reviewed)abstract
- Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB−/− mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1β, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP—protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.
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| 11. |
- Yao Mattisson, Ingrid, et al.
(author)
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Immune responses against oxidized LDL as possible targets for prevention of atherosclerosis in systemic lupus erythematosus
- 2021
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In: Vascular Pharmacology. - : Elsevier BV. - 1537-1891. ; 140
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Journal article (peer-reviewed)abstract
- Patients suffering from systemic lupus erythematosus (SLE) are at increased risk of developing cardiovascular disease (CVD) and traditional therapies including statins provide insufficient protection. Impaired removal of apoptotic material is a common pathogenic mechanism in both SLE and atherosclerosis and is considered to be a key factor in the development of autoimmunity. Since oxidized LDL and apoptotic material bind to the same receptors, we aimed to investigate if targeting the oxidized LDL autoimmunity can affect atherosclerosis in SLE. To investigate the possible role of oxidized LDL autoimmunity in the accelerated atherosclerosis associated with SLE we used a hypercholesterolemic SLE mouse model (B6.lpr.ApoE−/− mice). Promoting LDL tolerance through mucosal immunization with an apolipoprotein B-100 peptide p45 (amino acids 661–680) and cholera toxin B-subunit fusion protein increased regulatory T cells and B cells in mesenteric lymph nodes and reduced plaque development in the aorta by 33%. Treatment with the oxidized LDL-specific antibody Orticumab reduced aortic atherosclerosis by 43%, subvalvular plaque area by 50% and the macrophage content by 31%. The present study provides support for oxLDL as a possible target for prevention of cardiovascular complications in SLE. © 2021 The Author(s)
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| 12. |
- Zalem, Dani, et al.
(author)
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Characterization of the ganglioside recognition profile of Escherichia coli heat-labile enterotoxin LT-IIc
- 2022
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In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 32:5, s. 391-403
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Journal article (peer-reviewed)abstract
- 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 Neu5Ac alpha 3Gal or Neu5Gc alpha 3Gal, e.g. the gangliosides GM3, GD1a and Neu5Ac alpha 3-/Neu5Gc alpha 3--neolactotetraosylceramide and Neu5Ac alpha 3-/Neu5Gc alpha 3-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.
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