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Title
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Structural basis for the specific neutralization of Stx2a with a camelid single domain antibody fragment
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Author
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Abstract
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| Background: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113-rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb113(2) exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb113(2) with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation. |
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Language
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English
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Source (journal)
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Toxins
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Publication
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2018
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ISSN
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2072-6651
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DOI
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10.3390/TOXINS10030108
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Volume/pages
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10
:3
(2018)
, 20 p.
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Article Reference
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UNSP 108
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ISI
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000428565500016
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Medium
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E-only publicatie
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Full text (Publisher's DOI)
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Full text (open access)
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