Abstract

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

Highlights

  • Botulinum neurotoxin (BoNT) is the etiological agent of the disease botulism

  • Botulism often arises as a food-borne disease; BoNT alone is not sufficient to cause it and non-toxic non-hemagglutinin (NTNH) and HA are required for oral toxicity

  • In the case of serotype B, NTNH potentiates the oral toxicity of BoNT by about twenty-fold compared with BoNT alone

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Summary

Introduction

Botulinum neurotoxin (BoNT) is the etiological agent of the disease botulism. Different types of this toxin are produced by various strains of the spore-forming anaerobic bacteria Clostridium botulinum, C. butyricum, and C. barati, and are conventionally classified into seven serotypes, designated A through G [1]. BoNTs exist as large protein complexes by associating with nontoxic proteins termed neurotoxin-associated proteins (NAPs), which consist of non-toxic non-hemagglutinin (NTNH) and hemagglutinin (HA) [4]. BoNT and NTNH form a protein complex termed 12S toxin or M toxin. In serotypes A–D and G, another complex termed 16S toxin or L toxin is formed, which consists of BoNT, NTNH, and HA. Type A toxin is reported to form 19S toxin, which is considered to be a dimer of 16S toxin [5]

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