Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2.

Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1.

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain HN, and the C-terminal receptor binding domain HCC are largely resolved, but that of the HCN domain sandwiched between HN and HCC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their HCN domains or swapping HCN domains between each other. Both deletion and replacement of TeNT HCN domain by HCNA and HCNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping HCN domains between BoNT/A and B hardly impaired their biological activity, but substitution with HCNT did. Binding assays revealed that in the absence of HCN, not all receptor binding sites are equally well accessible. In conclusion, the presence of HCN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the HCN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that HCNT plays a different role in the intoxication mechanism of TeNT.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins' light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A-F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A-F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.

Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins' high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3-80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.

Botulinum neurotoxin serotype D - A potential treatment alternative for BoNT/A and B non-responding patients.

OBJECTIVES: Botulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers. METHODS: BoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days. RESULTS: Despite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action. CONCLUSIONS: BoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs. SIGNIFICANCE: BoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1.

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.

Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58⁻K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities.

In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin's cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2 recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.09 Šresolution crystal structure of the light chain protease domain of BoNT/HA (LC/HA). Structural comparison between LC/HA and LC of BoNT/F1 (LC/F1) reveals distinctive hydrophobic and electrostatic features near the active sites, which may explain their different VAMP-2 cleavage sites. When compared to BoNT/F5 that cleaves VAMP-2 at the same site as BoNT/HA, LC/HA displays higher affinity for VAMP-2, which could be caused by their different surface charge properties surrounding a VAMP-2 exosite-binding cleft. Furthermore, systematic mutagenesis studies on VAMP-2 and structural modeling demonstrate that residues R47 to K59 spanning the cleavage site in VAMP-2 may adopt a novel extended conformation when interacting with LC/HA and LC/F5. Taken together, our structure provides new insights into substrate recognition of BoNT/HA and paves the way for rational design of small molecule or peptide inhibitors against LC/HA.

Novel in-situ lamella fabrication technique for in-situ TEM.

In-situ transmission electron microscopy is rapidly emerging as the premier technique for characterising materials in a dynamic state on the atomic scale. The most important aspect of in-situ studies is specimen preparation. Specimens must be electron transparent and representative of the material in its operational state, amongst others. Here, a novel fabrication technique for the facile preparation of lamellae for in-situ transmission electron microscopy experimentation using focused ion beam milling is developed. This method involves the use of rotating microgrippers during the lift-out procedure, as opposed to the traditional micromanipulator needle and platinum weld. Using rotating grippers, and a unique adhesive substance, lamellae are mounted onto a MEMS device for in-situ TEM annealing experiments. We demonstrate how this technique can be used to avoid platinum deposition as well as minimising damage to the MEMS device during the thinning process. Our technique is both a cost effective and readily implementable alternative to the current generation of preparation methods for in-situ liquid, electrical, mechanical and thermal experimentation within the TEM as well as traditional cross-sectional lamella preparation.

The hypothetical protein P47 of Clostridium botulinum E1 strain Beluga has a structural topology similar to bactericidal/permeability-increasing protein.

Botulinum neurotoxins (BoNTs) are causative agents of the life-threatening disease botulism. They are naturally produced by species of the bacteria Clostridium botulinum as stable and non-covalent complexes, in which the BoNT molecule is assembled with several auxiliary non-toxic proteins. Some BoNT serotypes, represented by the well-studied BoNT serotype A (BoNT/A), are produced by Clostridium strains that carry the ha gene cluster, which encodes four neurotoxin-associated proteins (NTNHA, HA17, HA33, and HA70) that play an important role to deliver and protect BoNTs in the gastrointestinal tract during oral intoxication. In contrast, BoNT/E- and BoNT/F-producing strains carry a distinct gene cluster that encodes five proteins (NTNHA, P47, OrfX1, OrfX2, and OrfX3, termed the orfX cluster). The structures and functions of these proteins remain largely unknown. Here, we report the crystal structure of P47 resolved at 2.8 Å resolution. Surprisingly, P47 displays a structural topology that is similar to bactericidal/permeability-increasing (BPI) like proteins, which were previously identified only in eukaryotes. The similarity of a hydrophobic cleft of P47 with the phospholipid-binding groove of BPI suggests that P47 might be involved in lipid association to exert its function. Consistently, P47 associates and induces aggregation of asolectin-containing liposomes in a protein- and lipid-concentration dependent manner. These findings laid the foundation for future structural and functional studies of the potential roles of P47 and OrfX proteins in facilitating oral intoxication of BoNTs.

A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding.

Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission.

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C α-51) and BoNT/C L200W/M221W/I226W (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.