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Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans but also are common therapies for neurological diseases. BoNTs are dichain toxins, comprising an N-terminal catalytic domain (LC) disulfide bond linked to a C-terminal heavy chain (HC) which includes a translocation domain (H N ) and a receptor binding domain (H C ). Recently, the BoNT serotype A (BoNT/A) subtypes A1 and A2 were reported to possess similar potencies but different rates of cellular intoxication and pathology in a mouse model of botulism. The current study measured H C A1 and H C A2 entry into rat primary neurons and cultured Neuro2A cells. We found that there were two sequential steps during the association of BoNT/A with neurons. The initial step was ganglioside dependent, while the subsequent step involved association with synaptic vesicles. H C A1 and H C A2 entered the same population of synaptic vesicles and entered cells at similar rates. The primary difference was that H C A2 had a higher degree of receptor occupancy for cells and neurons than HcA1. Thus, H C A2 and H C A1 share receptors and entry pathway but differ in their affinity for receptor. The initial interaction of H C A1 and H C A2 with neurons may contribute to the unique pathologies of BoNT/A1 and BoNT/A2 in mouse models.KEYWORDS botulinum toxin, gangliosides, synaptic vesicles, synaptic vesicle protein 2, TIRF microscopy, clostridial neurotoxins, Clostridium botulinum, toxins B otulinum neurotoxins (BoNTs) are AB exotoxins secreted by several species of the genus Clostridium. BoNTs are single-chain 150-kDa proteins cleaved by either bacterial or host proteases to a dichain comprising a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC) linked by an interchain disulfide bond. The HC includes a translocation domain (H N ) and receptor binding domain (H C ) (1). The H C includes an N-terminal subdomain (H CN ) of limited known function and a C-terminal subdomain (H CC ) that confers neuron specificity by binding dual neuron-specific receptors. There are seven BoNT serotypes (A to G) (2). BoNT serotype A (BoNT/A) binds a ganglioside and synaptic vesicle glycoprotein 2 (SV2) (3-6), which allows rapid toxin entry via synaptic vesicles. Acidification of the synaptic vesicle lumen triggers H N to form a channel to facilitate LC translocation into the cytosol. Intracellular LC cleaves a SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) protein (7-12). SNARE protein cleavage inhibits exocytosis of cholinergic synaptic vesicles at neuromuscular junctions. BoNT LCs are long-lived proteases, sustaining paralysis in humans for several months depending on the serotype (2), which led to the licensing of BoNT/A and BoNT/B for human therapies (13,14).BoNT serotypes include subtypes neutralized by serotype-specific antisera (15-17). Informatics has classified eight BoNT/A subtypes (A1 to A8), which vary by ϳ10 to 15% in amino acid identity (15,16,(18)(19)(20)(21). While BoNT/A1 and BoNT/A2 cleave SNAP25 with similar kinetics (22-2...

Subsets of protein toxins utilize gangliosides as host receptors. Gangliosides are preferred receptors due to their extracellular localization on the eukaryotic cell and due to their essential nature in host physiology. Glycosphingolipids, including gangliosides, are mediators of signal transduction within and between eukaryotic cells. Protein toxins possess AB structure-function organization, where the A domain encodes a catalytic function for the posttranslational modification of a host macromolecule, including proteins and nucleic acids, and a B domain, which encodes host receptor recognition, including proteins and glycosphingolipids, alone or in combination. Protein toxins use similar strategies to bind glycans by pockets and loops, generally employing hydrogen bonding and aromatic stacking to stabilize interactions with sugars. In some cases, glycan binding facilitates uptake, while in other cases, cross-linking or a second receptor is necessary to stimulate entry. The affinity that protein toxins have for host glycans is necessary for tissue targeting, but not always sufficient to cause disease. In addition to affinity for binding the glycan, the lipid moiety also plays an important role in productive uptake and tissue tropism. Upon endocytosis, the protein toxin must escape to another intracellular compartment or into cytosol to modify a host substrate, modulating host signaling, often resulting in cytotoxic or apoptotic events in the cell, and a unique morbidity for the organism. The study of protein toxins that utilize gangliosides as host receptors has illuminated numerous eukaryotic cellular processes, identified the basis for developing interventions to prevent disease through vaccines and control bacterial diseases through therapies. In addition, subsets of these protein toxins have been utilized as therapeutic agents to treat numerous human inflictions.

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HC) and a receptor binding domain (HC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC/A1 and HC/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >10-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1. M-BoNT/A1 vaccination protected against challenge by 10 LD Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1 vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1 surviving BoNT challenge had dominant antibody responses to the LCHC domain, but varied antibody responses to HC. Sera from mice vaccinated with M-BoNT/A1 also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1 elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.

The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation. IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.