Abstract
Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.
Highlights
Botulinum neurotoxin (BoNT) is one of the most toxic substances known in nature and classified as a Tier 1 select agent by the Centers for Disease Control and Prevention (CDC) in the UnitedStates [1,2,3]
We further show that the binding epitopes of these VHHs are conserved within known Botulinum neurotoxin serotype E (BoNT/E) subtypes, suggesting that these VHHs merit further evaluation as therapeutic agents for the treatment of BoNT/E intoxication
We recently obtained a large panel of unique BoNT/E1-binding VHHs and identified three VHHs, termed JLE-E5, JLE-E9, and JLE-G6, that neutralize BoNT/E1 intoxication through binding at unique epitopes [51]
Summary
Botulinum neurotoxin (BoNT) is one of the most toxic substances known in nature and classified as a Tier 1 select agent by the Centers for Disease Control and Prevention (CDC) in the UnitedStates [1,2,3]. There are at least seven immunologically distinct serotypes of BoNT (A–G) that include more than 40 subtypes [4,5,6,7,8]. BoNT/E is one of the four BoNT serotypes (with BoNT/A, BoNT/B, and rarely BoNT/F) known to cause human botulism. There are 12 BoNT/E subtypes (E1–12) identified. While the majority of BoNT/E intoxication cases are foodborne illnesses caused by consumption of contaminated fish or marine mammal products [13], wound and infant botulism have been reported [14,15]. BoNT/E is being developed in Phase II clinical trial for therapeutic and aesthetic applications, which increases the potential risk of human exposures to this toxin [16]
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