Searching genomic databases revealed a novel BoNT gene

In an attempt to survey the evolutionary landscape of BoNTs, we performed iterative Hidden Markov model searches of the Uniprot sequence database. Our search identified all known BoNT subtypes and mosaic toxins, as well as the related tetanus neurotoxin (Fig. 1a; Supplementary Fig. 1). To our surprise, the search revealed a potentially new BoNT, tentatively designated BoNT/X (Fig. 1a, GenBank no.: BAQ12790.1), from the recently reported genomic sequence of C. botulinum strain 111. BoNT/X showed the least protein sequence identity with the other BoNTs in pairwise comparisons (Fig. 1b). Furthermore, the low sequence similarity is evenly distributed along the entire BoNT/X sequence (Fig. 1c), indicating that it is not a mosaic toxin. Despite this low sequence identity, the overall domain arrangement of BoNTs is conserved in BoNT/X (Fig. 1c), including a zinc-dependent protease motif HEXXH (residues 227–231, HELVH) in the LC (ref. 33), and a SXWY motif in the H C (residues 1,274–1,277, SAWY), which recognizes the lipid receptor gangliosides34.

Figure 1: Identification of BoNT/X. (a) A phylogenetic split network covering all BoNT serotypes, subtypes, mosaic toxins and related tetanus neurotoxin (TeNT) illustrates their potential evolutionary relationships, as well as conflicts arising from e.g. chimerisms, based on their protein sequences. BoNT/X is highlighted in red. An enlarged version of this panel is shown in Supplementary Fig. 1, with the sequence access number for each toxin gene noted. (b) A phylogenic tree of the protein sequence alignment for BoNT/A-G, TeNT and BoNT/X, analysed by the ClustalW method. The percentages of sequence identity between each toxin and BoNT/X are noted. (c) Upper panel: a schematic drawing of the three domains of BoNT/X, with conserved protease motif in the LC and the ganglioside binding motif in the H C noted. Lower panel: analysis using a sliding sequence comparison window demonstrated that the low similarity between BoNT/X and other BoNTs/TeNT is evenly distributed along the entire BoNT/X sequence. The X axis represents the query sequence position at the center of a 100-amino-acid moving sequence-comparison window. The Y axis shows the percentage of identity between that sequence window and each of the aligned background sequences. The two bars at the top of the graph illustrate the best matching sequence (lower bar) and whether the best match is significantly separated from the second-best match (upper bar). (d) A schematic drawing of the orf gene cluster that hosts the BoNT/X gene (upper panel), which has two distinct features compared with other known orfX clusters (middle and lower panels): (1) there is an additional orfX2 protein (designated orfX2b) located next to the BoNT/X gene; (2) the reading frame of orfX genes has the same direction as the BoNT/X gene. Full size image

Similar to the other BoNTs, the BoNT/X gene is located in a gene cluster23. All seven established BoNTs are co-expressed with another 150 kDa protein known as NTNHA (non-toxic non-hemagglutinin protein), which forms a pH-dependent complex with BoNTs and protects them from proteases in the gastrointestinal tract35. The BoNT/X gene is also preceded by a potential NTNHA gene (Fig. 1d). Besides BoNT and NTNHA, a typical BoNT gene cluster contains genes encoding one of the two types of accessory proteins: (1) the HA cluster encoding three conserved proteins HA17, HA33 and HA70, which form a complex with BoNT/NTNHA and facilitate absorption of toxins across the intestinal epithelial barrier36,37,38; or (2) the OrfX cluster encoding conserved OrfX1, OrfX2, OrfX3 and P47 proteins with unknown function23. The BoNT/X gene is located in an OrfX gene cluster, as are BoNT/E, F and members of BoNT/A. Interestingly, the BoNT/X cluster has two unique features (Fig. 1d): (1) there is an additional OrfX2 gene that does not exist in any other BoNT clusters (we designated it OrfX2b); (2) the reading frame of OrfX genes is usually opposite to BoNT/NTNHA genes, but it has the same direction as the BoNT/X gene in the BoNT/X cluster (Fig. 1d). These findings suggest that BoNT/X is a unique branch of the BoNT family.

The LC of BoNT/X cleaves VAMP2 at a novel site

To characterize BoNT/X, we first focused on its LC (X-LC, residues 1–439) and produced it as a His6-tagged protein in Escherichia coli. LCs of BoNT/A (A-LC) and BoNT/B (B-LC) were produced and assayed in parallel as controls. Incubation of X-LC with rat brain detergent extracts (BDE) did not affect syntaxin 1 or SNAP-25, but abolished VAMP2 immunoblot signals (Fig. 2a). LCs of BoNTs are zinc-dependent proteases33. As expected, EDTA prevented cleavage of SNARE proteins by X-, A- and B-LCs (Fig. 2a). Furthermore, incubation of X-LC with the purified recombinant cytosolic domain of VAMP2 (residues 1–93) converted VAMP2 into two lower-molecular-weight bands (Fig. 2b), confirming that X-LC cleaves VAMP2.

Figure 2: The LC of BoNT/X cleaves VAMPs at a unique site. (a) X-LC was incubated with BDE. Immunoblot analysis was carried out to detect syntaxin 1, SNAP-25 and VAMP2. Synaptophysin (Syp) served as a loading control. A-LC and B-LC were analysed in parallel. Cleavage of VAMP2 by B-LC results in loss of immunoblot signals, while cleavage of SNAP-25 by A-LC generates a smaller fragment (marked with an asterisk). EDTA blocked the activity of X-, A- and B-LCs. (b) VAMP2 (1–93) was incubated with X-LC. Samples were analysed by SDS–PAGE and Coomassie Blue staining. X-LC converted VAMP2 (1–93) into two smaller fragments. (c–e) VAMP2 (1–93) was incubated with X-LC. Samples were analysed by mass spectrometry (LC–MS/MS) to determine the molecular weight of cleaved fragments. Eluted peptide peaks from the HPLC column are plotted over running time (RT, X axis). The mass spectrometry data for the two cleavage products are colour-coded, with mass-to-charge ratio (m/z) noted. The molecular weight is deduced by multiplying m with z, followed by subtracting z. The protein sequences for the two cleavage products are colour-coded and listed in c. (f) Sequence alignment between VAMP family members, with the cleavage sites for BoNT/B, D, F, G and X marked in red, and the two SNARE motifs in blue shade. (g) HA-tagged VAMP1, 3, 7 and 8, and Myc-tagged Sec22b and Ykt6 were expressed in 293T cells via transient transfection. Cell lysates were incubated with X-LC and subjected to immunoblot analysis. Actin is a loading control. (h) GST-tagged Ykt6 was incubated with X-LC (100 nM). Samples were analysed by SDS–PAGE and Coomassie Blue staining. (i) GST-tagged VAMP2 (33–86), VAMP4 (1–115) and VAMP5 (1–70) were incubated with X-LC (100 nM). Samples were analysed by SDS–PAGE and Coomassie Blue staining. X-LC cleaved both VAMP4 and VAMP5. We note that VAMP5 protein contains a contaminant band that runs close to the cleavage product. (j) Experiments were carried out as described in a, except that VAMP4 and Sec22b were detected. Synaptotagmin I (Syt I) is a loading control. X-LC cleaved native VAMP4 in BDE. One of two (b,g,j) or three (a,h,i) independent experiments is shown. Full size image

To identify the cleavage site, we analysed the VAMP2 (1–93) protein, with or without pre-incubation with X-LC, by liquid chromatography–tandem mass spectrometry (LC–MS/MS, Fig. 2c–e). A single dominant peptide peak appeared after incubation with X-LC (Fig. 2c,e; Supplementary Fig. 2). Its molecular weight is 3,081.7, which fits only the peptide sequence of A67-L93 of VAMP2 (Fig. 2c,e). Consistently, another fragment from the beginning of the His6-tag to residue R66 of VAMP2 was also detected (Fig. 2d). To further confirm this finding, we repeated the assay with a different VAMP2 fragment: glutathione S-transferase (GST) tagged VAMP2 (33–86) (Supplementary Fig. 3). Incubation with X-LC generated a single dominant peptide peak with a molecular weight of 2,063.1, which fits only A67-R86 of VAMP2 (Supplementary Fig. 3). Together, these results demonstrate that X-LC has a single cleavage site on VAMP2 between R66 and A67.

R66-A67 is a novel cleavage site on VAMP2, distinct from all established target sites of BoNTs (Fig. 2f). It is also the only BoNT cleavage site located within a region previously known as the SNARE motif (Fig. 2f, shaded regions)39. The VAMP protein family includes VAMP1, 2, 3, 4, 5, 7 and 8, as well as related Sec22b and Ykt6. R66-A67 is conserved in VAMP1 and 3, which are highly homologous to VAMP2. To validate the specificity of X-LC, we expressed HA-tagged VAMP1, 3, 7, 8 and Myc-tagged Sec22b and Ykt6 in HEK293 cells via transient transfection. Cell lysates were incubated with X-LC. Both VAMP1 and 3 were cleaved by X-LC, whereas VAMP7, VAMP8 and Sec22b were resistant to X-LC (Fig. 2g).

BoNT/X cleaves VAMP4, VAMP5 and Ykt6

Unexpectedly, Ykt6 was also cleaved by X-LC (Fig. 2g). This finding was confirmed using a purified GST-tagged Ykt6 fragment, which shifted to a lower-molecular-weight band after incubation with X-LC (Fig. 2h). The cleavage site was determined to be K173-S174 by mass spectrometry analysis of the intact Ykt6 versus Ykt6 cleaved by X-LC (Supplementary Fig. 4). This site is homologous to the cleavage site of BoNT/X on VAMP2 (Fig. 2f). Among VAMP family of proteins, VAMP4 contains the same pair of residues (K87-S88) at this site as Ykt6. We found that X-LC cleaved both purified GST-tagged cytoplasmic domain of VAMP4 (Fig. 2i), as well as native VAMP4 in BDE (Fig. 2j). As a control, Sec22b was not cleaved by X-LC in BDE. In addition, the GST-tagged cytoplasmic domain of VAMP5 was also cleaved (Fig. 2i). The cleavage sites were determined by mass spectrometry analysis to be K87-S88 in VAMP4 and R40-S41 in VAMP5 (Supplementary Fig. 5). Both sites are homologous to the cleavage site of BoNT/X on VAMP2 (Fig. 2f), demonstrating that the location of the cleavage site is conserved across different VAMPs. The ability of X-LC to cleave VAMP4, VAMP5 and Ykt6 is highly unusual, as their sequences are substantially different from VAMP1/2/3. BoNT/X is the first and the only BoNT known that can cleave VAMPs beyond the canonical targets VAMP1, 2 and 3 (ref. 40).

Proteolytic activation of BoNT/X

We next examined the linker region between the LC and the HC, which must be cleaved by bacterial or host proteases to convert the toxin to an ‘active’ di-chain form. We produced a recombinant X-LC-H N fragment (residues 1–891) in E. coli and subjected it to limited proteolysis by endoproteinase Lys-C. Samples were analysed using Tandem Mass Tag (TMT) labelling and tandem mass spectrometry. TMT labels free N-termini (and lysines). Limited proteolysis by Lys-C produced one new free N terminus mapped to residue N439 in the linker region (Fig. 3a; Supplementary Data 1), confirming that the linker region is susceptible to proteases.

Figure 3: Proteolytic activation and inter-chain disulfide bond in BoNT/X. (a) Sequence alignment of the linker between the LC and HC of the seven established BoNTs plus BoNT/X. The Lys-C cutting site was identified by mass spectrometry analysis (see Method and Supplementary Data 1). (b) Cultured rat cortical neurons were exposed to X-LC-H N for 12 h. Cell lysates were harvested and immunoblot analysis carried out to examine syntaxin 1, SNAP-25 and VAMP2. Actin is a loading control. Trypsin-activated A-LC-H N and B-LC-H N were analysed in parallel. X-LC-H N entered neurons and cleaved VAMP2. X-LC-H N activated by Lys-C showed a greater potency than non-activated X-LC-H N . X-LC-H N was more potent than B-LC-H N and A-LC-H N , neither of which cleaved their substrates. (c) WT and mutant X-LC-H N were activated by Lys-C and analysed by SDS–PAGE and Coomassie Blue staining, with or without DTT. C461S and C467S mutants showed as a single band at ∼100 kDa without DTT, and separated into two ∼50 kDa bands with DTT. A portion of WT X-LC-H N formed aggregates, marked by an asterisk, which disappeared with DTT. The majority of activated WT X-LC-H N separated into two ∼50 kDa bands without DTT. This is due to disulfide bond shuffling as described in the following panel. (d) Lys-C-activated WT X-LC-H N was incubated with NEM to block disulfide bond shuffling. Samples were then analysed by SDS–PAGE and Coomassie Blue staining. A majority of WT X-LC-H N exists as a single band at ∼100 kDa without DTT after NEM treatment, indicating that native WT X-LC-H N contains an inter-chain disulfide bond. (e) Schematic drawings of the disulfide bond in WT and three cysteine mutants of BoNT/X. (f) Experiments were carried out as described in b, except that neurons were exposed to WT or X-LC-H N mutants. C423S mutation abolished the activity of X-LC-H N , whereas mutating C461 or C467 did not affect the activity of X-LC-H N . These results confirmed that the inter-chain disulfide bond is essential for the activity of X-LC-H N , and this inter-chain disulfide bond can be formed via either C423-C461 or C423-C467. One of two (b) or three (b,c,f) independent experiments is shown. Full size image

We then examined whether proteolytic activation enhances the potency of BoNT/X. It has been shown that incubation of high concentrations of LC-H N of BoNTs with cultured neurons results in entry of LC-H N , likely through non-specific uptake into neurons41. Similarly, X-LC-H N entered cultured rat cortical neurons and cleaved VAMP2 in a concentration-dependent manner (Fig. 3b). Activation by Lys-C increased the potency of X-LC-H N : 10 nM activated X-LC-H N cleaved similar levels of VAMP2 as 150 nM intact X-LC-H N (Fig. 3b). Activated X-LC-H N appears to be more potent than activated LC-H N of BoNT/A (A-LC-H N ) and BoNT/B (B-LC-H N ), which did not show any detectable cleavage of their substrates under the same assay conditions (Fig. 3b).

The inter-chain disulfide bond in BoNT/X

Like other BoNTs, the linker region of BoNT/X contains two conserved cysteines, but there is also an additional cysteine (C461) unique to BoNT/X (Fig. 3a). To determine the cysteine residues that form the essential inter-chain disulfide bond, we generated three X-LC-H N mutants, each with one of the three cysteine residues mutated (C423S, C461S and C467S). These mutants, as well as wild-type (WT) X-LC-H N , were subjected to limited proteolysis with Lys-C and then analysed via SDS–PAGE and Coomassie Blue staining, with or without the reducing agent dithiothreitol (DTT; Fig. 3c). Mutating the only cysteine on the LC (C423S) is expected to abolish the inter-chain disulfide bond. Consistently, C423S mutant separated into two ∼50 kDa bands without DTT. In contrast, both C461S and C467S mutants showed as a single band at 100 kDa in the absence of DTT and separated into two ∼50 kDa bands in the presence of DTT. These results suggested that C423 on the LC can form the inter-chain disulfide bond with either C461 or C467 on the HC. We also found that Lys-C treatment degraded a significant portion of C423S mutant as compared with C461S or C467S mutants (Fig. 3c, +DTT), suggesting that losing the inter-chain disulfide bond makes the molecule more susceptible to proteases. We noticed that a portion of WT X-LC-H N formed aggregates at the top of the SDS–PAGE gel (Fig. 3c, marked by an asterisk). These aggregates disappeared in the presence of DTT. C423/C461/C467 are the only three cysteines in the X-LC-H N ; mutating any one of them abolished formation of aggregates (Fig. 3c, −DTT), suggesting that these aggregates are formed by inter-molecular disulfide bonds due to the existence of an extra cysteine in the linker region.

Interestingly, the majority of activated WT X-LC-H N separated into two ∼50 kDa bands without DTT (Fig. 3c), which is similar to C423S mutant. On the other hand, WT X-LC-H N did not show increased degradation by Lys-C compared with C423S mutant (Fig. 3c, +DTT). One possible explanation is that WT X-LC-H N contains an inter-chain disulfide bond under native conditions, but this bond can rearrange to intra-chain C461–C467 pair under denaturing conditions in the SDS buffer. This phenomenon is known as disulfide bond shuffling, which often occurs among adjacent cysteines. To test this hypothesis, we utilized an alkylating reagent, N-Ethylmaleimide (NEM), which permanently blocks free cysteines and prevents disulfide bond shuffling. As shown in Fig. 3d, WT X-LC-H N pretreated with NEM showed as a single band at 100 kDa in the absence of DTT, and separated into two ∼50 kDa bands in the presence of DTT. These results confirm that WT X-LC-H N contains mainly an inter-chain disulfide bond, but it is susceptible to disulfide bond shuffling due to an extra cysteine in the linker region (Fig. 3e).

We further examined the activity of the three X-LC-H N cysteine mutants on cultured neurons. As expected, C423S mutant was inactive, whereas C461S and C467S mutants both showed similar levels of activity as WT X-LC-H N (Fig. 3f). These results confirm that the inter-chain disulfide bond is critical for the activity of BoNT/X.

Generating full-length BoNT/X via sortase-mediated ligation

We then sought to determine whether full-length BoNT/X is a functional toxin. As no antisera against BoNT/X are available, we decided to avoid generating the full-length active toxin gene. Instead, we developed an approach to generate a limited amount of full-length BoNTs in test tubes by enzymatic ligation of two non-toxic fragments of BoNTs. This method utilizes a transpeptidase known as sortase42,43, which recognizes the peptide motif LPXTG, cleaves between T-G, and concurrently forms a new peptide bond with other proteins/peptides containing N-terminal glycine (Fig. 4a). We produced two non-toxic fragments of BoNT/X: (1) LC-H N with a LPETGG motif and a His6-tag fused to the C terminus; and (2) the H C of BoNT/X (X-H C ) with a GST tag, thrombin cleavage site, and an additional glycine residue at its N terminus. Cutting by thrombin releases X-H C with a free glycine at its N terminus. Incubation of these two fragments with sortase generated a small amount of ∼150 kD full-length BoNT/X (X-FL, Fig. 4a,b). We note that X-H C showed poor solubility and a strong tendency towards aggregation, which might be the reason for the low ligation efficiency (Fig. 4b). In contrast, ligation of X-LC-H N with the H C of BoNT/A (A-H C ) achieved a better efficiency, with the majority of X-LC-H N ligated into a XA chimeric toxin (Supplementary Fig. 6a). To ensure biosafety, the amount of precursor fragments in the reaction is strictly limited to generate the minimum amount of ligated toxin necessary for functional assays.

Figure 4: Full-length BoNT/X is active on cultured neurons and in vivo in mice. (a) A schematic drawing of the sortase ligation method. (b) Sortase ligation reaction mixtures were analysed by SDS–PAGE and Coomassie Blue staining. The asterisk marks the proteins aggregates due to inter-molecular disulfide bonds. Full-length BoNT/X (X-FL) appeared only in the sortase ligation mixture. (c) Neurons exposed to the sortase ligation mixture (15 μl) or control mixtures for 12 h in culture medium. Cell lysates were analysed by immunoblot. The mixture containing both X-LC-H N and X-H C (but not sortase) cleaved slightly more VAMP2 than X-LC-H N alone. Ligating X-LC-H N and X-H C by sortase further enhanced cleavage of VAMP2, demonstrating that ligated X-FL is functional on neurons. (d) BoNT/A-G, BoNT/DC and BoNT/X were subjected to the dot blot assay, using four horse antisera (trivalent anti-BoNT/A, B and E, anti-BoNT/C, anti-BoNT/DC and anti-BoNT/F), as well as two goat antisera (anti-BoNT/G and anti-BoNT/D). BoNT/X is composed of X-LC-H N and X-H C at 1:1 molar ratio. These antisera recognized their corresponding target toxins, yet none recognized BoNT/X. The antisera against BoNT/DC and BoNT/C cross-react, as these two toxins share a high degree of similarity within their H C domains. (e) Cultured rat cortical neurons were exposed to ligated X-FL in culture medium for 12 h, with or without two combinations of anti-sera. Ab1: trivalent anti-BoNT/A/B/E, anti-BoNT/C and anti-BoNT/F. Ab2: anti-BoNT/G and anti-BoNT/D. The trivalent anti-BoNT/A/B/E was used at 1:50 dilution. All other anti-sera were used at 1:100 dilution. None of the antisera affected the cleavage of VAMP2 and VAMP4 by X-FL. The specificity and potency of these antisera were validated for their ability to neutralize target serotypes in the same assay as described in Supplementary Fig. 7. (f) X-FL linked by sortase reaction (0.5 μg) was injected into the gastrocnemius muscles of the right hind limb of mice (n=4). The injected limb developed typical flaccid paralysis, and the toes failed to spread within 12 h. The left limb was not injected with toxins, serving as a control. (g) Full-length inactive form of BoNT/X (BoNT/X RY ) was purified as a His6-tagged recombinant protein in E. coli. Further purified BoNT/X RY is shown in Supplementary Fig. 8b. One of two (e) or three (c,d) independent experiments is shown. Full size image

We first analysed the activity of ligated BoNT/X using cultured rat cortical neurons. Neurons were exposed to the sortase ligation mixture and control mixtures in culture medium. As shown in Fig. 4c, X-LC-H N alone cleaved some VAMP2 due to its high concentration in the reaction mixture. Mixing X-H C with X-LC-H N without sortase slightly enhanced cleavage of VAMP2 compared with X-LC-H N alone, suggesting that X-H C might be associated with X-LC-H N via non-covalent interactions. This interaction appears to be specific, as mixing A-H C with X-LC-H N did not enhance cleavage of VAMP2 in neurons (Supplementary Fig. 6b). Ligating X-LC-H N with X-H C by sortase clearly enhanced cleavage of VAMP2 compared with the mixture of X-LC-H N and X-H C without sortase (Fig. 4c). These results demonstrated that the X-H C is functional for targeting cells and that ligated full-length BoNT/X entered neurons and cleaved VAMP2. Similarly, ligated XA also entered neurons and cleaved VAMP2 (Supplementary Fig. 6b).

BoNT/X was not recognized by antisera against known BoNTs

We next carried out dot blot assays using antisera raised against known BoNTs, including all seven serotypes as well as one mosaic toxin (BoNT/DC), to confirm that BoNT/X is serologically unique. Four horse antisera were utilized (trivalent anti-BoNT/A, B and E, anti-BoNT/C, anti-BoNT/DC, and anti-BoNT/F) as well as two goat antisera (anti-BoNT/G and anti-BoNT/D). The specificity and potency of these antisera were first validated by analysing their ability to neutralize BoNTs on cultured neurons. As expected, all antisera neutralized their target BoNTs, without affecting the activity of a different serotype (Supplementary Fig. 7). We found that these antisera recognized their corresponding BoNTs in the dot blot assay, yet none recognized BoNT/X (Fig. 4d).

We further analysed whether the toxicity of BoNT/X on neurons can be neutralized by these antisera. X-FL generated by sortase-mediated ligation was first activated with limited proteolysis using trypsin. We used trypsin to activate X-FL instead of Lys-C for functional assays, as trypsin allows us to stop proteolysis using trypsin inhibitors. Activated X-FL entered cultured rat cortical neurons and cleaved both VAMP2 and VAMP4 in a concentration-dependent manner (Fig. 4e). Combinations of antisera against known BoNTs (Ab1 (horse antisera): trivalent anti-BoNT/A, B and E, anti-BoNT/C, and anti-BoNT/F; Ab2 (goat antisera): anti-BoNT/G and anti-BoNT/D) did not affect the activity of ligated X-FL, as evidenced by similar degrees of VAMP2 and VAMP4 cleavage in the presence of these antisera (Fig. 4e). These results confirmed that BoNT/X is a new BoNT serotype.

BoNT/X induced flaccid paralysis in vivo in mice

We next sought to determine whether BoNT/X is active in vivo using a well-established non-lethal assay in mice, known as the Digit Abduction Score (DAS) assay, which measures local muscle paralysis following injection of BoNTs into mouse hind limb muscles44. BoNTs cause flaccid paralysis of limb muscles, which is manifested as the failure to spread the toes in response to a startle stimulus. We injected ligated X-FL (0.5 μg, activated by trypsin treatment) into the gastrocnemius muscles of the right hind limb in mice, which induced typical flaccid paralysis and the failure of toes to spread (Fig. 4f), indicating that BoNT/X is capable of causing flaccid paralysis in vivo. We note that the potency of ligated X-FL appears to be much lower than other BoNTs in this assay. To further confirm the low toxicity of ligated X-FL, we injected mice with 1 μg of ligated X-FL intraperitoneally (n=3). No mice showed any systemic effects and all survived at this dose. Thus, ligated X-FL has a rather low toxicity in vivo in mice compared with other native BoNTs, which usually have lethal doses at low picogram levels per mouse.

Full-length inactive BoNT/X