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.Peer reviewe

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain

AbstractBotulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

AbstractBotulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties

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

The translocation domain (HN) of Botulinum neurotoxins (BoNTs) mediates the delivery of the BoNT light chain (LC) into neuronal cytosol. Here the authors provide insights into HN membrane insertion by determining the crystal structure of BoNT/A1 HN at acidic pH, which reveals a molecular switch in HN, where buried α-helices are transformed into surface-exposed hydrophobic β-hairpins

Super-resolving botulinum neurotoxin type A molecules: from surface landing to internalization in synaptic vesicles

Botulinum neurotoxin type-A (BoNT/A) is internalized into motor nerve terminals as part of its intoxication strategy to incapacitate nerve-muscle communication. The “dual receptor” model explained how BoNT/A initially interacts with GT1b gangliosides, thereby concentrating the toxin on the presynaptic membrane to foster the subsequent interaction with a proteinaceous co-receptor SV2 which triggers receptor-mediated endocytosis. I will revisit the “dual receptor” concept using two single-molecule imaging strategies,1-3 allowing tracking of single Atto647N-labeled BoNT/A molecules upon (i) landing on the plasma membrane (by uPAINT) and (ii) internalization in synaptic vesicles (by sdTIM) of living mature hippocampal neurons. With 30 to 40 nm localization precision, we revealed that once internalized in synaptic vesicles, Atto647N-tagged BoNT/A exhibits a markedly lower mobility than on the plasma membrane. I will discuss how individual genetic inactivation of the GT1b and SV2 binding sites of the neurotoxin affects the diffusion states of BoNT/A mutants on the plasma membrane and axonal trafficking. Single neurotoxin super-resolution imaging uncovers an updated dual receptor model taking into consideration the diffusive patterns generated by each of the co-receptors and leading to defined nanoscale dynamic organizations at key steps of the intoxication journey

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

Archive

<p>Soleus muscles of mice treated with BoNT/C-wt and used for the analysis of EJPs of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.g005" target="_blank">Fig 5B</a> were fixed immediately after the electrophysiological recordings and stained for <b>(A)</b> cleaved SNAP-25 (SNAP-25_c) or <b>(B)</b> Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.</p

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

<div><p>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 <i>S51T/R52N/N53P</i> (BoNT/C α-51) and BoNT/C <i>L200W/M221W/I226W</i> (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 <i>in vivo</i>. 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.</p></div

Time course of neuroparalysis recovery upon a local injection of BoNT/C variants in the mouse hind limb.

<p><b>(A)</b> Digit Abduction Score (DAS) assay. 1 LD₅₀ of BoNT/C-wt (cyan), or BoNT/C α-51 (green) or BoNT/C α-3W (red) were injected intramuscularly in the mice hind limb and neuroparalysis was evaluated according to [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref049" target="_blank">49</a>]. The rescue from paralysis was monitored daily until complete recovery was attained. Traces are representative of three independent experiments with at least 5 mice per condition. Error bars represent SEM <b>(B)</b> Analysis of evoked post synaptic junction potentials (EJP) on injected soleus muscles. Mice were treated as in A and at indicated time points soleus muscles were collected and processed for recordings of EJPs, as previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref027" target="_blank">27</a>]. Data are presented as a percentage of EJPs of control muscles. Each point represents an average EJP amplitude obtained from at least 45 muscle fibers from three different mice per condition. Statistical significance at each time point was determined by a Student's t-test comparing the mean values between either BoNT/C α-51 (green) or BoNT/C α-3W (red) compared to BoNT/C-wt (cyan) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. not significant). Error bars represent SEM.</p

BoNT/C mutants display noticeable lower potency than wild type BoNT/C.

<p><b>(A)</b> Activity of BoNT/C variants at the MPN hemidiaphragm assay. The black trace represents a dose-response calibration curve reporting the T₅₀ value obtained at indicated bath concentration of a reference wild type BoNT/C [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.ref045" target="_blank">45</a>]. Recombinant BoNT/C-wt (black diamond), tested at 100 pM displays a T₅₀ comparable to the previous BoNT/C-wt used at the same concentration. BoNT/C α-3W (white square) and BoNT/C α-51 (black square) need much higher concentrations to achieve a T₅₀ within the calibration curve. Error bars represent SD of n = 3–4 technical replicates. <b>(B)</b> Calculation of potency of BoNT/C mutants employing a power function fitted to the dose-response calibration curve in A. <b>(C)</b> Immunofluorescent analysis of hemidiaphragms derived from MPN assays. Hemidiaphragms treated with the indicated toxin and concentration were fixed immediately upon completion of paralysis and stained for cleaved SNAP-25 (SNAP-25_c, red). NMJs were spotted with α-Bungarotoxin (α-BTX, in green). Images shown are representative of at least three independent experiments. Scale bar, 10 μm.</p