Entry of Clostridial Neurotoxins

The Clostridial Neurotoxins (CNTs), Botulinum Neurotoxins (BoNTs) and Tetanus Neurotoxin (TeNT), are regarded as the most potent toxins for humans. They are secreted exotoxins from clostridial species and cause lethal paralysis in a matter of days. Tetanus is introduced by puncture wounds and is characterized by spastic paralysis and deregulation of the autonomic and sympathetic nervous systems; a toxoid-based vaccine has been widely used and provides efficacious protection. Botulism is a disease of flaccid paralysis usually acquired by food poisoning. BoNT toxoid vaccines are less effective: botulism is caused by seven immunologically distinct serotypes, and toxoid vaccines are less stable. However, BoNTs have been used as treatments for spastic muscle disorders since the early 1990s. Due to the rarity of disease and the therapeutic potential, vaccines have been limited to at-risk individuals and military personnel. Current research aims to develop better botulism vaccines, evaluate serotypes for clinical use, and develop therapeutic platforms for treating other diseases. This body of work focuses on identifying unique molecular mechanisms of BoNT entry by lesser characterized serotypes and subtypes: BoNT/C, BoNT/D, and BoNT/A2.;BoNTs and TeNT are 150 kDa AB exotoxins cleaved into a di-chain molecule of 50 kDa and 100 kDa subunits. The smaller protein, termed the light chain (LC), is a zinc protease that cleaves SNARE proteins of neurons and inhibits exocytosis of neurotransmitters. The larger protein, termed the heavy chain, is divided into two subunits: the translocation domain (HCT) and the receptor binding domain (HCR). The HCR binds and enters neurons, and the HCT delivers the LC across the endosomal membrane into the neuronal cytosol in order to reach its target. Generally, the neurotoxins are not cytotoxic on the cellular level, but yet lethal to birds and mammals due to respiratory musculature paralysis. TeNT targets neurons of the central nervous system and reaches target cells by trafficking through peripheral a-motor neurons, whereas BoNT intoxication remains primarily peripheral. The pathology difference of TeNT and BoNTs is due to cellular trafficking and targeting.;BoNTs target a-motor neurons by binding to dual receptors, both of which are specifically expressed by neurons. The primary receptor is a ganglioside: a glycosphingolipid anchored to the outer plasma membrane by a ceramide group with a four-carbohydrate backbone of glucose--galactose--N-Acetylgalactosamine--galactose, decorated with varying numbers of sialic acid residues. The most prominent ganglioside expressed by neurons is GT1b, which has three sialic acids. BoNT/A binds GT1b by the terminal galactose-sialic acid moiety. BoNT/B, /E, /F, and /G contain similar ganglioside binding pockets (GBPs). The secondary receptor is a synaptic vesicle protein, either synaptic vesicle glycoprotein (SV2) or synaptotagmin. Synaptic vesicle proteins are rapidly endocytosed after neurotransmitter release, facilitating uptake of bound BoNTs.;When this work began, the crystal structures for HCR/C and HCR/D were unknown, but the ganglioside binding regions were noted to be different than other BoNT serotypes due to sequence dissimilarity. Furthermore, interactions between BoNT/C and synaptic vesicle proteins could not be demonstrated under conditions where BoNT/A and BoNT/B interacted with their respective receptors. In the first two chapters of this work, using the crystal structures of HCR/C and HCR/D solved by the Kim laboratory, the receptor binding capabilities were explored. Both HCR/C and HCR/D have unique specificity to b-series gangliosides, and it is hypothesized that both interact with two carbohydrate receptors instead of a ganglioside and a protein. However, HCR/D retains specificity for entry by the synaptic vesicle pathway, but the entry pathway of HCR/C is yet unclear. These toxins have greater similarity to TeNT, which enters neurons by binding two gangliosides, and raise questions about CNT cellular targeting. The latter work in Chapter 4 characterizes the HCR of the BoNT/A2 subtype, which is 89% identical to the prototype HCR/A1, but enters neurons more efficiently. In animal models, this leads to a quicker onset of paralysis with a lower dosage. While ganglioside affinity was proposed to be responsible for this phenotypic difference, we demonstrated that HCR/A2 is evolutionarily optimized to enter more efficiently by contributions from the N-terminal half of the HCR, which is hypothesized to contain receptor binding properties.;From this work, diversity among BoNT serotypes and subtypes was demonstrated. While all serotypes are lethal toxins, the specific mechanisms by which BoNTs cause disease are varied. Characterization of lesser-studied serotypes and newly identified subtypes may expand the therapeutic range of BoNTs in future work.