| Pathogenesis and Host Response | Research Article

Botulinum neurotoxin X lacks potency in mice and in
human neurons
Brieana M. Gregg,1 Takuhiro Matsumura,2 Travis G. Wentz,1 William H. Tepp,1 Marite Bradshaw,1 Pål Stenmark,3 Eric A. Johnson,1
Yukako Fujinaga,2 Sabine Pellett1

ABSTRACT Botulinum neurotoxins (BoNTs) are a class of toxins produced by Clostridium
botulinum (C. botulinum) and other species of Clostridia. BoNT/X is a putative novel
botulinum neurotoxin identified through genome sequencing and capable of SNARE
cleavage, but its neurotoxic potential in humans and vertebrates remained unclear.
The C. botulinum strain producing BoNT/X, Strain 111, encodes both a plasmid-borne
bont/b2 as well as the chromosomal putative bont/x. This study utilized C. botulinum
Strain 111 from Japan as well as recombinantly produced full-length BoNT/X to more
fully analyze this putative pathogenic toxin. We confirmed production of full-length,
catalytically active native BoNT/X by C. botulinum Strain 111, produced as a disulfidebonded dichain polypeptide similar to other BoNTs. Both the purified native and therecombinant BoNT/X had high enzymatic activity in vitro but displayed very low potency
in human-induced pluripotent stem cell-derived neuronal cells and in mice. Intraperito­
neal injection of up to 50 µg of native BoNT/X in mice did not result in botulism; however,mild local paralysis was observed after injection of 2 μg into the gastrocnemius muscle.
We further demonstrate that the lack of toxicity by BoNT/X is due to inefficient neuronalcell association and entry, which can be rescued by replacing the receptor binding
domain of BoNT/X with that of BoNT/A. These data demonstrate that BoNT/X is not apotent vertebrate neurotoxin like the classical seven serotypes of BoNTs.

IMPORTANCE The family of botulinum neurotoxins comprises the most potent toxins
known to humankind. New members of this family of protein toxins as well as moredistantly related homologs are being identified. The discovery of BoNT/X via bioinfor­
matic screen in 2017 as a putative new BoNT serotype raised concern about its potential
as a pathogenic agent with no available countermeasures. This study for the first timeassessed both recombinantly produced and native purified BoNT/X for its vertebrate
neurotoxicity.
KEYWORDS

botulinum neurotoxin, BoNT, Clostridium botulinum, BoNT/X

B

otulinum neurotoxins (BoNTs) are a family of the most potent biological toxins
known to humans and vertebrates and are produced by anerobic, Gram-positive,
spore-forming species of Clostridia (1–3). BoNTs are the causative agent of botulism,
a potentially lethal paralytic disease, characterized by persistent descending flaccid
paralysis (4, 5). The BoNT protein family is genetically diverse, consisting of at least sevendistinct antigenic serotypes (BoNT/A-G) with 37%–70% pairwise amino acid identity andover 40 subtypes (6). In terms of function, serotypes and subtypes can differ by neuronalcleavage target, cell entry, potency, and longevity of paralytic effect (6–10).
BoNTs are 150-kDa proteins composed of three major functional domains. After
expression as a 150-kDa polypeptide, BoNTs undergo post-translational proteolytic
activation via environmental or host proteases into a disulfide-linked ~50-kDa light
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Editor Richard Gerald Brennan, Duke University
School of Medicine, USA
Address correspondence to Yukako Fujinaga,
yukafuji@med.kanazawa-u.ac.jp, or Sabine Pellett,sabine.pellett@wisc.edu.
Brieana M. Gregg and Takuhiro Matsumura
contributed equally to this article. Author order wasdetermined alphabetically.
P.S. is an inventor on patents regarding BoNT/X. The
authors declare no additional conflict of interest.
See the funding table on p. 18.
Received 15 November 2023
Accepted 16 January 2024
Published 13 February 2024
Copyright © 2024 Gregg et al. This is an open-accessarticle distributed under the terms of the Creative
Commons Attribution 4.0 International license.
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AUTHOR AFFILIATIONS See affiliation list on p. 18.

chain (LC) and ~100-kDa heavy chain (HC) dichain protein (4, 6). The dichain pro­teins specifically target human and vertebrate neurons. Intoxication occurs primarily
at cholinergic nerve terminals, where the HC C-terminal domain (HC) binds specificganglioside and protein receptors on the neuronal cell surface, resulting in endocytosisof the holotoxin (5, 11). The N-terminal HC domain (HN) then facilitates translocationof the LC into the cytosol, where the LC is released from the HC by disulfide bond
reduction (12) and cleaves SNARE (soluble N-ethylmaleimide-sensitive factor attachment
protein receptors) proteins. SNARE proteins are an essential component of the exocyto­
sis machinery, and the specific cleavage by BoNTs disables this function and thereby
prevents fusion of acetylcholine-containing vesicles to the membrane of presynaptic
nerve terminal (13, 14). Absence of acetylcholine signaling in the synaptic cleft results inthe persistent flaccid muscular paralysis characteristic of botulism.
Formal serotype classifications (BoNT/A-G) are based on the absence of cross-neu­
tralization by antisera derived from a given serotype (15), although some variants
include chimeric BoNTs or novel BoNTs identified by DNA sequencing only, without
toxicity analyses of the entire toxin protein, defying simple classification (16). Increased
availability of bacterial genomes through whole genome sequencing has identified
non-clostridial BoNT homologs which continue to be discovered. These bioinformati­
cally discovered BoNT homologs and BoNTs share key motifs related to metallopro­tease function and a similar multi-domain structure. Briefly, BoNT/Wo (Weissella oryzae),
Cp1-3 (Chryseobacterium piperi), BoNT/Ef (Enterrococcus faecium), PMP1 (Paraclostridium
bifermentas), and PGT1/2 (Paeniclostridium ghonii) putative toxin genes were discovered
via bioinformatic screens, and their natural target cell types and species are unknown(17–20) . Some BoNT-like toxins have proven SNARE targets outside of vertebrates, suchas PMP1 which cleaved mosquito-derived syntaxin and was toxic to mosquito larvae
when expressed recombinantly (21).
In contrast to most BoNTs, originally discovered through their association withbotulism, the pathogenic potential of bioinformatically discovered BoNT homologs
remains unknown without active investigation. While studies have generally beenquick to demonstrate the enzymatic activity of the light chain against neuronal SNARE
substrates, many unknowns remain regarding the toxicity and activity of the full
holotoxin. Such studies are complicated by the regulatory restrictions in place for the
production and characterization of native and recombinant BoNTs found in Clostridium.
While SNARE cleavage and structural similarities link BoNTs and many BoNT
homologs, an important distinguishing factor is the production of the homologs by
non-clostridial species, whereas classical BoNTs A-G are expressed by C. botulinum
species. A unique exception among homologs is BoNT/X, a bioinformatically identified
BoNT with unknown toxicity discovered in C. botulinum strain, which is genetically moreclosely related to the non-clostridial homologs than to BoNT/A-G (22). BoNT/X shares~30% protein sequence identity with other BoNT serotypes but retains conservation at
key motifs and the broad domain structure of BoNTs (23). The bont/x was discovered in
the genome of C. botulinum Strain 111 in 2017, which prior to the discovery of bont/x
was considered a BoNT/B-producing strain isolated from a 1995 case of infant botulism
in Japan (24, 25). Antisera raised against BoNT/B was sufficient to ablate toxicity, andanalyses of patient samples taken over the course of recovery indicated that the strain
could naturally lose the bont/B2 gene, developing a non-toxigenic phenotype (26). Thisfinding was replicated by a laboratory study wherein serial passage of Strain 111 led toloss of the bont/B2 containing plasmid and loss of toxicity (27). The entire genome of
C. botulinum Strain 111 was sequenced in 2015 , and a 2017 study found that while thebont/b gene was encoded on a large plasmid, the strain possessed an additional putative
bont gene cluster on the chromosome, which was named bont/X (23). The bont/x genecluster contains NTNH and p47/orfX accessory genes arranged in a unique orientation
relative to those generally observed in bont/a, e, and f. These variations further suggestthat the bont/x gene cluster is significantly distant from the classical bont genes (16, 28).

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Functional analysis of recombinant BoNT/X LC expressed in E. coli revealed that
BoNT/X LC is a metalloprotease that cleaves Vesicle-associated Membrane Proteins(VAMP) 1, 2, 3, 4, and 5, as well as the SNARE homolog Ykt6 (23, 29). In vitro, the
BoNT/X LC displayed ~10-fold higher VAMP1 cleavage efficiency when compared with
the BoNT/B LC (29). However, this high potency in vitro did not translate to high potency
in vivo. A recombinant BoNT/X holotoxin was created by sortase ligation of E.coli-pro­
duced LC-HN and HC, and while intramuscular (i.m.) injection of up to 0.5 µg BoNT/X permouse resulted in local hindlimb paralysis, intraperitoneal (i.p.) injection of 1 µg BoNT/X
per mouse resulted in no death or symptoms of botulism (23). These data are in stark
contrast to the mouse i.p. lethal dose 50 (LD50) values for BoNT/B1 of 0.21–0.50 ng/kg(30, 31). While sortase ligation is capable of linking two protein domains, this method
inherently introduces changes into the protein and should not be used to exclude
neurotoxicity of the native putative toxin.
BoNTs and other neurotoxins are defined by their ability to specifically enter neuronalcells and disrupt neurotransmitter release, and while BoNT/X was shown to possess theability for SNARE cleavage and thus neurotransmitter release disruption, neuronal cell
entry has not been demonstrated. Furthermore, the atoxic phenotype of plasmid-cured(PC) C. botulinum Strain 111 indicates either a lack of expression or stability of BoNT/X
or inability of the toxin to form its active dichain form or inability of BoNT/X LC to reachits target within the neuronal cell. In this study, we sought to further investigate thepotential toxicity of BoNT/X. We analyzed full-length BoNT/X produced recombinantly in
E. coli and C. botulinum and examined endogenous production of native BoNT/X by C.
botulinum Strain 111. Our data indicate that BoNT/X was expressed in Strain 111 as anactive dichain, and the toxin displayed high levels of catalytic activity in vitro but little
to no toxicity to mice in vivo. Similarly, recombinant BoNT/X produced in E. coli or atoxic
C. botulinum was able to cleave human VAMPs but displayed inefficient neuronal cell
entry into human-induced pluripotent stem cell (hiPSC)-derived neurons. Taken together,
these data suggest BoNT/X’s apparent lack of toxicity in mice and human neurons is due
to poor or no neuronal cell entry.

BoNT/X produced recombinantly in E. coli is catalytically active in vitro but
inefficiently cleaves VAMP2 in neuronal cells
Catalytically inactive BoNT/XRY expressed in Escherichia coli (E. coli) BL21 (DE3) was used
to inoculate and boost mice for production of anti-BoNT/X antiserum. The serum was
successfully able to detect the immunogen BoNT/XRY as well as recombinant BoNT/X
expressed in atoxic C. botulinum strain Hall A hyper tox- (Fig. S1).
Wild-type (WT) BoNT/X containing a C-terminal his tag (denoted as BoNT/Xwt-his6)
was expressed in E. coli BL21 (DE3) and purified via the IMAC-Ni2+ column. Analysis of
the purified rBoNT/X via western blot showed significant aggregation as evidenced by
the high-molecular weight smear above the 150-kDa toxin band in the non-reduced
sample. Furthermore, toxin treated with LysC or trypsin was detected as two primary
bands of ~100 kDa and ~50 kDa, regardless of whether the samples were reduced or
not (Fig. 1a), indicating failure of trypsin or LysC to convert the rBoNT/X to a dichain
form linked by a disulfide bond. Trypsinized rBoNT/X treated with N-ethylmaleimide toprevent disulfide bond formation did not show significantly different SDS-PAGE band
patterns for reduced or non-reduced samples. Therefore, this observed lack of a 150-kDa
band in the non-reduced samples may be the result of disulfide bond shuffling aspreviously proposed (23) or proteolytic degradation by the enzymes meant to activate
the toxin.
A previous study found that aggregation of a BoNT/X LC-HCN could be eliminated via
mutation to one of the three cysteines in the toxin linker region—C423, C461, or C467—
which is in support of the disulfide bind shuffling hypothesis where improper disulfide
bonding prevents proper intrachain disulfide bond formation (23). However, point
mutations C467A and C461A in full-length BoNT/XRY did not improve aggregation in ourMarch 2024 Volume 15

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FIG 1 Characterization of recombinant BoNT/X expressed in E. coli. (a) Coomassie stained SDS-PAGE gel of BoNT/X expressed in E. coli BL21 (DE3), purified
activity of rBoNT/X in hiPSC-derived neuronal cell lysates. Cell lysates were incubated with either trypsinized or non-trypsinized BoNT/X in reducing buffer andwere analyzed via western blot for VAMP2 cleavage. Syntaxin was used as a loading control. (c) In situ activity of rBoNT/X in hiPSC-derived neurons. Cultured
neurons were exposed for 24 hours to serial dilutions of BoNT/X, and cell lysates were analyzed for VAMP2 cleavage via western blot. Estimated concentrations(nM) of BoNT/X are noted. (d) Antibody neutralization cell assay. Trypsin-treated BoNT/X (20 nM) was incubated with anti-BoNT/X antiserum for 1 hour, andhiPSC-derived neurons were exposed to the antibody-BoNT/X mixture, BoNT/X alone, anti-BoNT/X antiserum alone, or culture media alone for 40 hours. Cell
lysates were analyzed for VAMP2 cleavage via western blot, using syntaxin as a loading control.

un-reduced samples nor did it result in an observable change to the trypsin-cleavage
pattern between the mutants and wild-type BoNT/XRY (Fig. S3).
In a hiPSC-derived neuronal cell lysate assay (32), reduced rBoNT/X holotoxin
efficiently cleaved VAMP2 and trypsin treatment of the toxin increased cleavage activity
by approximately fivefold (estimated EC50 0.8 nM) when compared with non-trypsintreated samples (estimated EC50 0.16 nM) (Fig. 1b). These data are in agreement withprevious data showing the high enzymatic activity of BoNT/X LC (23, 29). Interestingly,
this high level of enzymatic activity of rBoNT/X was not observed in cell-based assays
using hiPSC-derived neurons, which requires cell entry of the toxin prior to endopepti­
dase activity (Fig. 1c). Here, serial dilutions of trypsin-treated and non-treated rBoNT/X
were only observed to cleave VAMP2 at concentrations greater than 3.8–15 nM, withtrypsinized toxin increasing catalytic activity, indicating activation by trypsin treatment.
Pre-incubation of trypsin-treated rBoNT/X with polyclonal anti-BoNT/X antiserum before
exposure to hiPSC-derived neurons completely abolished VAMP2 cleavage, confirmingthat the cleavage was specific to BoNT/X activity (Fig. 1d). It has been shown that the
BoNT/A LC-HN alone can enter neurons at concentrations greater than 10 nM and cleave
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via IMAC-Ni2+ column, and treated with LysC or trypsin at 250:1 (wt/wt) BoNT/X:LysC or trypsin. R, reduced (with 100 mM DTT); NR, non-reduced. (b) In vitro

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SNAP-25 (33); therefore, we cannot exclude the possibility that the observed VAMP2
cleavage by rBoNT/X at high nanomolar concentrations may be due to the non-specificuptake of rBoNT/X or fragments containing the LC rather than targeted translocation into
the neuronal cytosol.
Lastly, E. coli-derived rBoNT/X was injected i.p. into mice (n = 2) at concentrationsof ~30 µg/mouse non-trypsin treated and ~15 µg/mouse trypsin treated. Mice wereobserved for the following 48 hours for symptoms indicative of neuromuscular paralysis.
Mice injected with trypsin-treated rBoNT/X displayed no symptoms, although mice
injected with non-trypsin-treated samples exhibited mild symptoms but did not die.
In a Digit Abduction Score (DAS) Assay, mice (n = 2) were injected in the gastrocnemius
muscle with ~0.4 µg/mouse trypsin-treated or untreated rBoNT/X and the flacciditymediated reduction in digit spread was analyzed (34). There was no detectable paralysis(DAS = 0). Taken together, these data analyzing rBoNT/X produced in E.coli indicate little
to no neurotoxicity. However, since the rBoNT/X aggregated and was not converted toa disulfide-linked LC-HC dichain, low activity due to protein solubility and stability could
not be excluded. The lack of activity after trypsin treatment is likely related to the failure
to convert rBoNT/X to a disulfide-linked toxin dichain.

To produce stable rBoNT/X that undergoes normal post-translational processing,wild-type full-length BoNT/X was expressed in an atoxic Clostridium botulinum expres­sion host, strain Hall A hyper tox- (35, 36).
The rBoNT/X was produced as a 150-kDa un-nicked protein in this clostridial strain
within 24 hours, with apparent partial degradation ( Fig. S1). Further incubation resulted
in loss of the rBoNT/X, indicating that BoNT/X is unstable in culture supernatants ofproteolytic C. botulinum and that the proteasome of C. botulinum strain Hall A hyper
tox- does not convert BoNT/X to its dichain form. A small percentage of soluble BoNT/X
was recovered in the extract supernatant post-sonication while the majority of the toxin
remained associated with the cell pellet. A portion of the extract supernatant containing
soluble BoNT/X was kept for further analysis, while the remaining supernatant under­
went partial purification via SE (S-300) and IEX (DEAE) chromatography.
Serial dilutions of trypsin-treated or untreated rBoNT/X extract supernatant (Fig. 2)
and partial purification pool (data not shown) were exposed to hiPSC-derived neurons
for 72 hours, and cell lysates were examined for VAMP2 cleavage as described previously(32). Only a mild partial decrease of the VAMP2 band was observed, regardless of trypsin
treatment, indicating little VAMP2 cleavage. These data indicate that, similar to E.coliproduced rBoNT/X, rBoNT/X produced in an endogenous expression host also has little
to no neurotoxic activity. However, even after production in an endogenous expression
host, there was a lack of normal post-translational processing to the dichain form, andpartial degradation of the toxin was apparent. Mice (n = 4) were injected with 500 µL oftrypsin-treated or non-treated crude BoNT/X extract or partially purified BoNT/X DEAE
pool. Based on densitometry, the estimated injection concentration for the non-treated
rBoNT/X extract and rBoNT/X DEAE pool was 2.5 µg/mouse. Mice injected with BoNT/X
extract displayed initial mild distress, and mice injected with partially purified BoNT/X
from DEAE chromatography showed moderate signs of distress (labored breathing,lethargy) immediately after injection, but both groups quickly recovered and displayed
no further symptoms of botulinal intoxication throughout the remaining observation
period. The quick onset of initial symptoms seen in both groups is not consistent withbotulinal intoxication.

C. botulinum Strain 111 endogenously expresses BoNT/X at similar levels to
BoNT/B and proteolytically processes native BoNT/X to its dichain form
Native BoNT/X was analyzed to definitively determine whether BoNT/X may have humanand vertebrate neurotoxicity. The native C. botulinum Strain 111 is a bivalent toxin strain
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BoNT/X produced recombinantly in atoxic C. botulinum poorly cleaves
VAMP2 in situ and is atoxic to miceResearch ArticlemBio

FIG 2 Characterization of recombinant BoNT/X expressed in C. botulinum. In situ activity of rBoNT/X extract in hiPSC-derived
neurons. Cultured neurons were exposed for 72 hours to serial dilutions of rBoNT/X, and neuronal cell lysates were analyzed

isolated from a clinical botulism case in Japan, which encodes both a chromosomal
bont/x and a plasmid-borne bont/b2 (23). Strain 111 has previously been shown to readilylose the bont/b2-containing plasmid via repeated culture passaging (27); therefore, toanalyze endogenous BoNT/X expression, C. botulinum Strain 111 was serially passaged
until the bont/b2-bearing plasmid was lost. Plasmid loss was confirmed via PCR using
primers recognizing the bont/b2 gene and the bont/x genes (data not shown) and by
western blots probing with anti-BoNT/B antibody (Fig. 3). Western blot probing with
anti-BoNT/X antiserum showed that BoNT/X is endogenously expressed in C. botulinum
Strain 111 at levels consistent with other BoNTs and similar to expression of BoNT/B2
in wild-type Strain 111 (Fig. 3). There was no apparent change in quantity of BoNT/X
between the WT and PC samples under a variety of tested growth conditions and media,
suggesting no loss in toxin expression after the plasmid-curing process. Furthermore,
native BoNT/X was fully converted to its dichain form, as indicated by the 100-kDa HC
and 50-kDa LC bands in the reduced samples on the western blot (Fig. 4).

Culture supernatant of plasmid-cured Strain 111 containing BoNT/X is nontoxic to mice
Culture supernatants of WT and PC samples of Strain 111 grown for 4 days in either CMM,
TPM, or TPGY at 30°C or 37°C were injected i.p. into mice (n = 2). Mice were observed fordeath or other motor impairment symptoms consistent with botulism over the following
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for VAMP2 cleavage via western blot. Estimated concentrations (nM) of BoNT/X are noted.

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FIG 3 Production of BoNT/X and BoNT/B by WT and PC C. botulinum Strain 111. Cultures were grown anaerobically at 30°C or 37°C in 5 mL of cooked meat
medium (CMM), tryptone peptone glucose yeast extract (TPGY), or toxin production media (TPM) for 4 days. Reduced (via 100 mM DTT) samples were analyzed
via SDS-PAGE with anti-BoNT/B antibody (a) or anti-BoNT/X antiserum (b). Fifty nanograms of BoNT/B1 toxin was used as a positive control for both. Where noted,
the BoNT/B holotoxin and LC weakly cross-reacted with the anti-BoNT/X antiserum. HC, heavy chain; LC, light chain; HC+LC, holotoxin.

7-day period. The majority of mice injected with WT Strain 111 died within 3 hours postinjection, and all died within 24 hours (Table 1). There were no deaths nor symptoms ofbotulism in any of the mice injected with PC Strain 111 (Table 1). As WT Strain 111 wasshown to endogenously express BoNT/B and BoNT/X at similar quantities (Fig. 3), the lackof toxicity of PC samples in the mouse bioassay (MBA) is further indication that BoNT/X is
not a potent vertebrate neurotoxin.

BoNT/X was purified from C. botulinum Strain 111 as the minimal progenitor toxin
complex (M-PTC, final concentration of 340 μg/mL), as attempts to dissociate BoNT/X
from its non-toxic non-hemagglutinin. (NTNH) protein by ion exchange chromatogra­
phy under alkaline conditions resulted in BoNT/X being degraded ( Fig.S4). SDS-PAGE
showed that the isolated BoNT/X M-PTC in the absence of DTT forms two distinct bands
at ~140 kDa and ~150 kDa (Fig. 4, left). The former is likely NTNH/X, as treatment with
DTT resulted in no change in band size, whereas the ~150-kDa band separated into
distinct 100-kDa and 50-kDa bands upon reduction with DTT, indicating the presence ofthe native BoNT/X as a LC-HC dichain in culture supernatant. The identity of the 150-kDa
and 100- and 50-kDa bands was further confirmed by western blot (Fig. 4 right).

TABLE 1 MBA of 4-day culture supernatants of WT or PC C. botulinum Strain 111

Media

Day 1 (alive/tested)

Day 7 (alive/tested)

CMM 30°C WT
CMM 30°C PC
CMM 37°C WT
CMM 37°C PC
TPM 30°C WT
TPM 30°C PC
TPM 37°C WT
TPM 37°C PC
TPGY 30°C WTTPGY 30°C PC
TPGY 37°C WTTPGY 37°C PC

0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2

0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2
0/2
2/2

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BoNT/X is post-translationally processed to a dichain form in C. botulinum
Strain 111

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FIG 4 BoNT/X purified from C. botulinum Strain 111. BoNT/X was purified from plasmid-cured Strain 111 cultured 4 days in
anti-BoNT/X antiserum (right). HC+LC, holotoxin; HC, heavy chain; LC, light chain.

These data represent the first confirmation that BoNT/X is expressed in its native
strain and that it is post-translationally processed by host strain proteases to its fully
active dichain form.

Endogenously produced BoNT/X displays high catalytic activity
Dilutions of reduced BoNT/X ranging from 0 to 50 nM alongside dilutions of reduced
BoNT/B1 (Okra) were exposed to the extracellular domain of GST human VAMP2 in
an endopeptidase assay (Fig. 5a) (37). Analysis of the VAMP2 substrate via western
blot confirmed that, as with recombinantly expressed BoNT/X, endogenous BoNT/X ishighly catalytically active and displays ~10-fold higher cleavage than BoNT/B1. To further
confirm that the observed VAMP2 cleavage is due to catalytic activity of the BoNT/X LC,
BoNT/X was treated with the chelating agent ethylenediaminetetraacetic (EDTA) before
repeating the VAMP2 endopeptidase assay.
This resulted in a lack of cleavage in pre-treated BoNT/X samples (Fig. 5b). Addi­
tionally, preincubation of 5 nM BoNT/X with either mouse anti-BoNT/X antiserum or
anti-BoNT/B antibodies demonstrated that BoNT/X cleavage of VAMP2 is inhibited byanti-BoNT/X antiserum but unaffected by anti-BoNT/B antibodies (Fig. 5c). This suggests
BoNT/X is an active metalloprotease whose activity can be suppressed through theaddition of a chelating agent or anti-BoNT/X antiserum.

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TPGY. Reduced (R) and non-reduced (NR) samples were analyzed via SDS-PAGE gel (left) or SDS-PAGE probed with mouseResearch ArticlemBio

FIG 5 In vitro activity of native BoNT/X. (a) Western blot showing in vitro GST-human VAMP2 (2-94) cleavage by reduced BoNT/X and BoNT/B1 (Okra). The toxins
were exposed to VAMP2 for 45 minutes before analysis. (b) Western blot of in vitro cleavage of GST-human VAMP2 with 5 nM reduced BoNT/X or 50 nM reduced
BoNT/B either pre-incubated with 100 mM EDTA for 30 minutes (+) or are left untreated (−). (c) Western blot showing inhibition of GST-human VAMP2 cleavage
by 5 nM reduced BoNT/X if pre-incubated for 30 minutes with 1 μg anti-BoNT/X antiserum (+Anti-X) but not with 1 μg anti-BoNT/B rabbit polyclonal antibody(+Anti-B).

BoNT/X does not bind the PC12 cellular membrane
Rat pheochromocytoma PC12 cells were exposed to 300 nM BoNT/X or BoNT/B for 1
hour before fluorescent imaging in order to assess the ability of BoNT/X to bind theneuronal cell membrane (Fig. 6c). In addition to fluorescent antibodies targeting BoNT/X
and BoNT/B (green), antibodies binding DNA (Hoechst, blue) and F-actin (Phalloidin, red)
were used. There was no observed binding of BoNT/X to the cell membrane.

In vivo toxicity of the native BoNT/X M-PTC was tested via MBA. Mice (n = 3) were injected
i.p. with an estimated 5 µg/mouse or 50 µg/mouse BoNT/X M-PTC and monitored for
symptoms indicative of botulism or death during the following 4 days (Table 2). No micedisplayed symptoms nor did any die after injection. In a DAS assay (n = 3 mice), the left
hindlimb was injected i.m. with 2 µg of BoNT/X, and the right hindlimb was injectedwith an equivalent volume of vehicle alone. In control mice, 10 pg of BoNT/B (Okra) was
similarly injected alongside the vehicle. As shown in Fig. 6a, mice injected with 2 µg
BoNT/X and 10 pg BoNT/B both displayed a reduction in reflexive digit spread 24 hours
post-injection, although the average DAS score for mice injected with BoNT/X remained
lower than BoNT/B over the course of 5 days (Fig. 6b).

Chimeric BoNT/(X/A1)
To further explore BoNT/X non-toxicity, a recombinant X(LC-HCN)/A(HCC) [denoted as
BoNT/(X/A1)] chimera was expressed in E. coli, affinity purified, and trypsinized/non-tryp­sinized. BoNT/(X/A1) ( Fig. S5) was serially diluted from 200 ng to 20 pg and injected intomice to determine a MLD (Fig. 6d) . MLDs were 200 ng for the trypsinized and 2 ng forthe non-trypsinized BoNT/(X/A1), indicating the X LC-HCN is toxic to mice upon properdelivery to the motor neuron. Similar to E. coli-produced BoNT/X holotoxin, trypsin
treatment of BoNT/(X/A1) resulted in 100-kDa and 50-kDa bands on SDS-PAGE gels
under both reduced and non-reduced conditions, indicating unlinked dichain formation(Fig. S5). The data confirm that the trypsin-treated, E. coli-produced rBoNT/X fails toresult in a disulfide-linked dichain. Overall, these results are consistent with a previous
report (23) showing greater activity of a sortase-linked BoNT/X(LC-HCN)/A(HCC) chimera
TABLE 2 Mouse Bioassay of native BoNT/X isolated from plasmid-cured Strain 111

BoNT/X

Day 1 (alive/tested)

Day 4 (alive/tested)

5 µg/mouse
50 µg/mouse

3/3
3/3

3/3
3/3

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FIG 6 Native BoNT/X causes mild local paralytic effects on mice. (a) Mice (n = 3) were injected into the left gastrocnemius muscle with either 2.0 μg BoNT/X

average values and standard deviations depicted in the graph (n = 3). (c) Confocal microscopy depicting the lack of native BoNT/X binding to neuronal PC12 (rat)cells. (d) Minimal lethal dose (MLD) of recombinant X(LC-HCN)/A(HCC) chimera, expressed in E. coli. Estimated concentration BoNT/(X/A1) holotoxin is indicatedbased on densitometry of total purified protein.

in cultured neurons and further indicate that replacement of the HCC domain of BoNT/X
with HCC/A1 increases toxicity.

DISCUSSION
The discovery of BoNT/X via bioinformatic screen in 2017 as a proposed new BoNT
serotype raised concerns about its potential as a pathogenic agent with no availa­
ble countermeasures (23). Initial experiments suggested that recombinant BoNT/X LC
displayed higher cleavage efficiency than BoNT/B in vitro (23, 29). In addition, sortaseligated BoNT/X holotoxin exhibited some toxicity in mice, causing mild local paralysis
after injection of 0.5 µg into the gastrocnemius muscle, but no systemic symptoms
after intraperitoneal injection (23, 29). These data, however, could not exclude thepotential toxicity of native BoNT/X, since recombinantly expressed and enzymatically
ligated BoNT/X may differ in toxicity from the native toxin due to potential insufficient
processing of the toxin within the recombinant expression system or as a consequenceof conformational changes to BoNT/X following the sortase linking reaction. In addition
to these data, no information was available on whether the native C. botulinum Strain
111 produces full-length, stable BoNT/X in detectable quantities. As a result of thisMarch 2024 Volume 15

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complex or 10 pg BoNT/B (Okra). The right hindlimb was injected with vehicle. Shown are representative images of digital abduction at 24 hours post-injection,
where both BoNT/X and BoNT/B complexes result in moderate muscle paralysis. (b) Reduction in reflexive spread of digits was quantified via the DAS, with

potential toxicity, BoNT/X was classified as a Tier 1 Select Agent alongside BoNT
serotypes A-G.
This study for the first time assessed both recombinantly produced and native
purified BoNT/X for its potential as a vertebrate neurotoxin. Purified native and rBoNT/X
were tested for enzymatic activity in vitro, for cell entry and enzymatic function inside
cells in neuronal cells, and for full biologic activity in mice. Our results further provide
the first confirmation that BoNT/X is expressed by C. botulinum Strain 111 at levels
comparable to BoNT/B (Fig. 3). Importantly, while disulfide-bonded dichain formation
was not observed for rBoNT/X either by our group (Fig. S1) or by others (23, 38), native
BoNT/X was proteolytically processed into a ~150-kDa active dichain in Strain 111, as
evidenced by reduction of the 150-kDa toxin to the 100-kDa and 50 kDa HC and LC (Fig.
4).
C. botulinum Strain 111 is a dual-toxin-producing strain, with the bont/x localized on
the chromosome and a bont/b2 localized on a plasmid. Previous analysis of this strain
revealed an atoxic phenotype upon plasmid curing (26, 27), which led to the conclusionthat bont/x is either silent or BoNT/X is non-toxic. Analysis of the production of native
BoNT/X in either the C. botulinum Strain 111 wild type or the plasmid cured strain
unambiguously showed strong expression of BoNT/X (Fig. 3). These data suggest thatnative BoNT/X, as produced by its native strain, is not toxic to vertebrates or humans.
In an in vitro endopeptidase assay, native BoNT/X holotoxin cleaved VAMP2 more
efficiently than BoNT/B at the same concentrations (Fig. 5a), confirming previous studies
demonstrating the high catalytic activity of rBoNT/X LC (23, 29, 38). Furthermore, the
SNARE cleavage after exposure to BoNT/X holotoxin could be halted either by preincu­
bation with anti-BoNT/X antiserum (Fig. 1d and 5c) or EDTA (Fig. 5b), indicating theobserved cleavage is specific to the metalloproteolytic activity of BoNT/X LC. Similarly,
full-length rBoNT/X resulted in sensitive VAMP2 cleavage, which was still observable withestimated EC50 values of ~0.8 nM and ~0.15 nM for non-trypsin-treated and trypsin-trea­
ted samples, respectively (Fig. 1b, EC50 values quantitated from duplicate assays). This
compares to EC50 values of ~2 nM for BoNT/F1 and ~16 nM for BoNT/B1 in the sameassay.
With confirmation that BoNT/X is expressed endogenously in C. botulinum Strain
111 and is both capable and highly efficient at cleaving mammalian neuronal SNARES
in vitro, the question of the cause of the apparent lack of in vivo toxicity of plasmid
cured Strain 111 remains. Both purified native and rBoNT/X were examined for theirability to cause SNARE cleavage inside cultured neurons. In hiPSC-derived neuronal cells,
rBoNT/X either did not cleave VAMP2 (Fig. 2), or only partially cleaved VAMP2 at highnM concentrations (Fig. 1c). Since at nanomolar concentrations, non-specific entry andcleavage by LC or LC-containing fragments of the BoNT cannot be ruled out, these data
indicate low or no activity of rBoNT/X in cultured human neurons, which have previously
been shown to be exquisitely sensitive to BoNT/A, capable of detecting fM amounts (32).
One possible explanation for the lack of neuronal toxicity may be BoNT/X holotoxin’s
inability to efficiently bind and initiate translocation of the LC into neuronal cytosol.
An initial step of BoNT intoxication involves binding of the toxin HCC to gangliosides
and protein receptors on the surface of the neuron (39, 40). The classical serotypes
BoNT/A-G exhibit varying binding affinities to the most common mammalian neuronal
gangliosides GT1b, GM1a, GD1a, and GD1b (41, 42). However, a preprint manuscriptby Martínez-Carranza et al. found that rBoNT/X holotoxin displayed very weak binding
against these four gangliosides in vitro (38). In our own study, endogenously expressed
BoNT/X failed to bind neuronal PC12 cells (Fig. 6c), whereas BoNT/B sequestered to theneuronal cell membrane as expected.
Investigations of potential botulism-like toxicity of recombinant and native BoNT
further indicated the absence of any systemic toxicity and only very mild local paralysis
at very high concentration. Zhang et al. (23) previously reported that i.m. injection of
0.5 µg/mouse of sortase-linked rBoNT/X LC-HN induced local paralysis in mice. Interest­
ingly, we did not observe any detectable hindlimb paralysis (DAS = 0) following i.m.
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injection of ~0.4 µg/mouse rBoNT/X holotoxin isolated from E. coli. The low activity orlack of local paralysis may be in part due to the aggregation and instability of rBoNT/X,
which was present throughout the purification process, and failure to form the more
active disulfide-bonded dichain (Fig. 1a). However, 2 µg of native BoNT/X , which purified
as a disulfide-bonded dichain molecule (Fig. 4), did produce mild paralysis in mice
(average DAS = 2 at 24 hours post-injection) (Fig. 6a and b). This paralysis compares toa stronger paralysis caused by injection of 10 pg/mouse of BoNT/B1, suggesting thatnative BoNT/X requires over 200,000-fold more toxin to produce local paralysis in vivo
than BoNT/B. Interestingly, mice injected intraperitoneally with up to ~50 µg native
BoNT/X, ~30 µg rBoNT/X (expressed in E. coli), or ~2.5 µg rBoNT/X (expressed in C.
botulinum Hall A-hyper tox-) did not result in any mouse deaths. This indicates thatnative BoNT/X is at least 10 million-fold less toxic than BoNT/A in mice when injected
intraperitoneally. The lack of neuronal cell association in the PC12 cell model (Fig. 6c)and the high enzymatic activity of the LC indicate that the lack of toxicity is due to a
failure of BoNT/X to enter human or vertebrate neurons. In fact, replacement of the HCc
domain of rBoNT/X with the HCc domain of BoNT/A1 resulted in systemic toxicity in mice
(Fig. 6d), demonstrating that the translocation domain and LC of BoNT/X are functional
neurotoxin domains, while the receptor binding domain fails to specifically recognize
and bind to neuronal cells.
It is interesting to speculate that BoNT/X’s apparent lack of toxicity to mammalian
neurons is the result of the toxin targeting another cell type or perhaps another class ofvertebrate or non-vertebrate organisms. Although BoNT/X is expressed by a Clostridium
species and was therefore proposed to be a novel BoNT, it genetically groups moreclosely with the newly emerging class of non-clostridial BoNT homologs, many of whichcurrently have undefined target species (23). PMP1 is one exception in this lineage, as therecombinant toxin has been shown to cleave neuronal SNARES in Anopheles mosquito
larvae (21). As BoNT/X shares more protein sequence identity with PMP1 than the sevenclassic BoNT serotypes, it is possible that identifying BoNT/X’s target cell type may
require looking outside of vertebrate neurons. Considering that C. botulinum Strain 111
is producing a second, functional BoNT/B toxin, it is also possible that bont/x is a defunctgene in this strain that is maintained due to its incorporation into the chromosome
and lack of direct targeting of bont-gene clusters by endogenous CRISPR systems of C.
botulinum (43).
This study sought to clarify whether the proposed novel BoNT/X exhibits toxicity
comparable to other BoNTs and poses a threat to human health and safety. Whether
expressed in the native C. botulinum Strain 111 or recombinantly in C. botulinum or E.
coli, our data show that BoNT/X is not a potent vertebrate neurotoxin. Furthermore, thesedata indicate that the lack of toxicity by rBoNT/X is due to a combination of failure ofneuronal cell binding and a failure to form a disulfide-bonded dichain. However, thenative BoNT/X was converted to a disulfide-bonded dichain and remained unable tocause systemic botulism in mice at 50 µg, suggesting that the lack of toxicity of native
BoNT/X is primarily due to the lack of neuronal cell binding and entry. Determining
preferred receptor targets for BoNT/X binding will be an important next step in further
understanding the utility of these toxins to the bacteria expressing them and may inform
potential uses of BoNT/X for pharmaceutical or research applications.

MATERIALS AND METHODS
Our Wisconsin laboratory and personnel are registered with the CDC Federal SelectAgent Program for research involving botulinum neurotoxins and botulinum neurotoxinproducing strains of clostridia. The research program, procedures, occupational healthplan, documentation, security, and facilities are closely monitored by the University of
Wisconsin—Madison Biosecurity Task Force, University of Wisconsin—Madison Office of
Biological Safety, the University of Wisconsin—Madison DURC subcommittee, and atregular intervals by the CDC and the Animal and Plant Health Inspection Service (APHIS)
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as part of the University of Wisconsin—Madison Select Agent Program. All person­
nel have undergone suitability assessments and completed rigorous and continuing
biosafety training, including biosafety level 3 (BSL3) and select agent practices before
participating in laboratory studies involving botulinum neurotoxins and neurotoxi­
genic C. botulinum strains. All recombinant DNA protocols for the construction of
the recombinant BoNT genes and their expression in C. botulinum strains have been
approved by the University of Wisconsin Institutional Biosafety Committee (IBC), andspecific experiments were approved by the Federal Select Agent Program. A dual-use
research of concern (DURC) risk mitigation plan has been established and approvedby the University of Wisconsin—Madison DURC Subcommittee and NIAID for theseexperiments. Animal studies involving BoNT select agents were approved by the
University of Wisconsin—Madison IACUC.
Our laboratory in Kanazawa University has the permission to possess and handle
Clostridium botulinum and botulinum neurotoxins (Class II pathogens) by the Ministerof Health, Labor and Welfare of Japan, pursuant to the provisions of the Cabinet Orderof Japan. The research program and engaged persons are also authorized and closely
monitored by the Kanazawa University Safety Committee for pathogen. Animal studies
were conducted under the applicable laws and guidelines for the care and use oflaboratory animals at the Kanazawa University. They were approved by the Animal
Experiment Committee of the Kanazawa University (AP-214252).

Culture media reagents were purchased from Sigma-Aldrich (St. Louis, MO), BD Difco(Franklin Lakes, NJ), and Fujifilm (Osaka, Japan). Unless otherwise noted, antibodies forwestern blot analysis were produced in the lab (in the case of mouse-anti BoNT/X andrabbit-anti BoNT/B polyclonal antibodies) or purchased from Santa Cruz Biotechnology
Inc. (Dallas, TX) and Jackson ImmunoResearch (West Grove, PA). Antibiotics (chloramphe­
nicol, cycloserine, kanamycin, and thiamphenicol) were purchased from Sigma-Aldrichand used at the following concentrations: in E. coli, kanamycin 50 µg/mL, chloramphe­
nicol 25 µg/mL in agar, and 12.5 µg/mL in liquid media and C. botulinum, cycloserine
250 µg/mL and thiamphenicol 15 µg/mL.
E. coli was grown at 37°C in either liquid LB agitated at 225 rpm or on LB agar plates
supplemented with corresponding antibiotics for plasmid selection. E. coli 10β and BL21
(DE3) were purchased from New England Biolabs (Ipswich, MA), and the donor strain E.
coli CA434 was kindly provided by N. Minton (University of Nottingham, UK).
C. botulinum cultures were grown in static nitrogen-flushed hungate tubes, andhandling of culture was carried out within an anaerobic chamber (Forma Anerobic
System, Marietta, OH; atmosphere consisted of 80% N2, 10% CO2, and 10% H2). Larger
quantities of culture for purification were grown statically in aerobic conditions. Cultures
were grown in the indicated liquid media or agar plate formulated as follows: TPGY)—
5% Trypticase peptone, 0.5% Bacto peptone, 0.4% glucose, 2% yeast extract, and 0.1%
L-cysteine HCl (pH 7.3–7.4); TPM—2% Trypticase peptone, 0.5% glucose, and 1% yeast
extract (pH 7.3); peptone yeast glucose media (PYG)—2% proteose peptone, 0.5% yeast
extract, 0.5% glucose, and 0.025% sodium thioglycolate, pH 7.0; CMM—0.1 g/mL H2O
supplemented with 0.3% glucose and 0.2% soluble starch.
C. botulinum Hall A hyper tox- (35) is an atoxic, non-sporulating C. botulinum
expression host. Using Clostron (36), an intron was introduced into the A1 toxin geneand confirmed via PFGE and southern blotting. Western blotting and mouse bioassay
further confirmed the lack of toxin production.

Production of BoNT/X-specific polyclonal antibody
Catalytically inactive BoNT/X (BoNT/XRY) was produced in E. coli BL21 (DE3) essentiallyas described previously (23). E. coli cultures were grown in a shaker flask instead ofa bioreactor, and after induction, cells were incubated for 19 hours and lysed using
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a French press. Culture lysate was clarified by centrifugation and passed through a
5-mL HisPur Ni-NTA agarose (Thermo, Waltham MA, #88222) column to produce purified
BoNT/XRY. Four female ICR mice were injected with 20 µg/mouse of the catalyticallyinactive holotoxin in 0.4 mL of alhydrogel intraperitoneal and 0.1 mL subcutaneousand boosted in the same manner after 2 weeks. Seven days post-boost, the mice werebled via the maxillary vein and serum was prepared. Specificity of the mouse serum
was tested by western blot and was successfully able to detect recombinant BoNT/X
expressed in C. botulinum Hall A hyper tox-.

Plasmid curing of C. botulinum Strain 111
Glycerol stock of C. botulinum Strain 111 (Osaka Prefecture University) was anaerobicallycultured in 10 mL (PYG) media for 3 days at 37°C. Whole culture was passaged into fresh
media every 3 days to cure the strain of the bont/b2-containing plasmid. For verificationof plasmid loss, serially passaged culture was spread onto Brucella HK plates and grownanaerobically at 37°C for 24 hours. Single colonies were then grown anaerobically in 5 mL
CMM at 37°C. After day 3, aliquots of cultures were centrifuged 5,000 g for 10 minutesat room temperature and serotype-specific (BoNT/B) PCR was performed to confirm theloss of the plasmid (data not shown). Western blots further confirmed the loss of thebont/b2-containing plasmid from Strain 111. One sample each of the WT and PC colonies
verified by serotype-specific PCR were grown up to 4 days in CMM, TPM, or TPGY
liquid media. Samples were grown at either 30°C or 37°C, and whole culture aliquotsof each were taken at days 1 and 4. The anti-BoNT/B western blot primary antibody
was anti-BONT/B1 Rb/PC, and the secondary antibody was anti-rabbit IgG HRP. The
anti-BoNT/X western blot primary antibody was BoNT/X-specific polyclonal antiserum
produced in mice, and the secondary antibody was anti-mouse IgG HRP. BoNT/B1 toxin
control in both western blots was 50 ng BoNT/B1.

Expression and purification of rBoNT/X
The gene for catalytically inactive BoNT/X was encoded on the E. coli expression plasmid
pET22b. This gene was altered via site-directed mutagenesis to the catalytically active
form by converting amino acids A360R and F363Y. This resulted in wild-type BoNT/X
which was his-tagged on the C-terminus (denoted as BoNT/Xwt-his6) and expressedin E. coli BL21 (DE3). Cells were lysed by sonication on ice in 80 mL of 50 mm Hepes
buffer pH 7.2 containing 500 mM NaCl, 45 mM imidazole, 20 mg lysozyme, protease andphosphatase inhibitor (Pierce A32962), and deoxyribonuclease I (Sigma-Aldrich, D5025).
The concentrated extract was passed through a 5-mL HisPur Ni-NTA agarose (Thermo
88222) column, and bound BoNT/X was eluted with sonication buffer containing 250 mM
imidazole. Purified BoNT/X was treated with trypsin at ~350:1 (wt:wt) toxin:trypsin or
toxin:lysC for 15 minutes at room temperature. Non- trypsin-treated and trypsin-treated
BoNT/X samples were then reduced via DTT or not reduced before analysis by SDS-PAGE
and western Blotting.

C. botulinum hall A hyper toxThe gene for catalytically active BoNT/X was cloned into a pMTL8000 series shuttle
vector pMTL83152 (44), transformed into the E. coli conjugative donor strain CA434,
and conjugated into C. botulinum Hall A hyper tox- as previously described (45, 46).
After mating, cells were resuspended and plated onto fresh media supplemented with
15 µg/ml thiamphenicol (selection for vector plasmid) and 250 µg/mL cycloserine(selection for C. botulinum). Transconjugant colonies were selected at random andscreened for the presence of the expression vector via plasmid isolation and restriction
digest, and for toxin production via western blot.

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A 6-L static culture of Hall A-hyper/tox− (rBoNT/X) was grown 24 hours at 37°C inliquid TPGY supplemented with 15 µg/mL thiamphenicol. Cells from the 24-hour culture
were collected by centrifugation (7,500 g, 10°C, 5 minutes) and stored at −80°C for
further processing. Cell pellets were removed from −80°C, thawed, and evenly suspen­
ded in 40 mL of sonication/extraction buffer [200 mM sodium/potassium phosphatebuffer pH 6.0 containing 10% glycerol, 20 mM DTT, 1.5 mg RNAse A, 2 mg DNAse, andprotease inhibitor cocktail (Pierce #A32922)]. The suspension was sonicated for 3 minutes
in total on time (10 sec on/30 sec off) at 70% amplitude on ice. For SE chromatography,
the suspension was centrifuged at 15,000 g for 20 minutes at 10°C. The supernatant wassequentially filtered through 0.45-micron and 0.22-micron filters and the filtered extract
applied to a Sephacryl S-300 (Cytiva, Marlborough, MA) column 2.5 × 91 cm equilibrated
with 25 mM sodium citrate buffer pH 5.5 containing 10 mM DTT. The column was washedovernight with pH 5.5 citrate buffer containing DTT at room temperature. The opticaldensity of fractions was measured, and peak fractions were analyzed by SDS-PAGE andwestern blot. Fractions containing BoNT/X were pooled and precipitated with ammo­nium sulfate (39 g/100 mL). For DEAE chromatography pH 5.5, the precipitate from the
S-300 pool was collected by centrifugation (12,000 g, 4°C, 20 minutes) and the pellet
dissolved in 4 mL of 50 mM sodium citrate buffer pH 5.5 containing 50 mM DTT. The
solution was centrifuged at 12,000 g, 20°C, for 15 minutes, and the clarified supernatant
was applied to a DEAE sephadex A50 column 2.5 × 18 cm equilibrated with 50 mM
sodium citrate pH 5.5 + 50 mM DTT. Fractions were analyzed by SDS-PAGE and western
blot for the presence of BoNT/X. Fractions from the first peak containing the majority of
BoNT/X were pooled and precipitated with ammonium sulfate (39 g/100 mL and stored
at 4°C).

C. botulinum Strain 111 cured of the plasmid containing the bont/b2 gene was inoculated
into liquid TPGY media and grown statically at 30°C for 4 days. The culture supernatant
was ammonium sulfate (60%) precipitated and stored overnight at 4°C. Precipitate wascollected by centrifugation (10,000 g, 4°C, 20 minutes), and the pellet was solubilized
in 50 mM sodium phosphate buffer pH 6.0. RNAse A (Sigma Aldrich) was added (0.1 mg/mL), and the solution was incubated at 37°C for 6 hours. The digest was centrifuged at
12,000 g, at room temperature for 20 minutes, and the supernatant was precipitated withammonium sulfate (60% saturation) and stored at 4°C. The precipitated material wascollected by centrifugation (12,000 g, 4C, 30 min) and solubilized in 50 mM sodium
phosphate buffer pH 6.0 containing 200 mM NaCl. The solution was dialyzed andconcentrated with a 10-kDa molecular weight cutoff (MWCO) Amicon spin concentrator
to a final volume of 5 mL. The solution was sequentially filtered through 0.45-micron and0.22-micron filters before size exclusion chromatography on a HiLoad 16/600 Superdex
column. Fractions containing BoNT/X were pooled and dialyzed against 50 mM sodium
citrate buffer pH 5.0 and then applied to a cation exchange column (HiTrap SP HP(GE Healthcare:Cytiva)] and eluted with a sodium chloride gradient (0–1M). Fractions
containing BoNT/X were dialyzed against 25 mM sodium phosphate buffer pH 7.4 andapplied to a DEAE FF (Cytiva) column. Bound BoNT/X was eluted with a NaCl gradient(0–1M), and fractions containing the toxin were concentrated with an Amicon 100-kDa
MWCO spin concentrator for analysis.

Expression and purification of recombinant BoNT/X-A1 chimera
rBoNT/A (CAL82360.1) and rBoNT/X (BAQ12790.1) were synthesized as E. coli codonoptimized gblocks (IDT, San Diego, CA) consisting of the light chain and translocation
domains (LC-HCN) of BoNT/X and binding domain (HCC) of BoNT/A1. The gBlocks were
blunt ligated into pJET1.2 vectors (Thermo Fisher Scientific), PCR amplified via Phusion
Hot Start Flex (New England Biolabs, Ipswich, MA) with overlap primers, assembled
into a NdeI, XhoI linearized pET-15b vector in a three-fragment Hifi assembly (NEB) in
fusion with the thrombin cleavable N-terminal his6 tag, and transformed into DH10B andMarch 2024 Volume 15

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then BL21 DE3 competent E. coli (New England Biolabs, Ipswich, MA). Whole plasmid
sequencing via nanopore sequencing at ≥1,000× coverage was utilized to sequence
verify the assembled construct.
To survey expression of the rBoNT/(X/A1)-pET-15b construct in Bl21, a 200-mL
expression culture (LB + 100 µg/mL carbenicillin) was inoculated with overnight culture,grown at 37°C, 225 RPM, induced to 1 mM IPTG at OD600 0.4–0.6, and expressed for
24–27.5 hours at 16°C, 225 RPM. Whole cell lysate was evaluated for expression via
SDS-PAGE Coomassie staining or western blot with 1° polyclonal α-A1 or α-X antibody
or monoclonal α-His antibody (BioLegend 906102). Expression culture was pelleted at
6,000 × g for 8 minutes, resuspended in 50 mL sonication buffer [50 mM Tris, 50 mM
NaCl, 20 mM imidazole, 5% glycerol, and protease inhibitor cocktail tablet (Pierce), pH5.5], sonicated and loaded on to an equilibrated 2–3-mL Ni-IMAC column, washed tobackground with the same buffer, and eluted with 250 mM imidazole.
Peak NiNTA purified fractions of rBoNT/(X/A1) were trypsin treated at 1:50 trypsin
at 37°C for 30 minutes. To acquire a rough minimum lethal dose for rBoNT/X/A1, NT/
T-treated semi-purified rBoNT/X/A1 was serially diluted in 50 mM Tris, 50 mM NaCl at pH
5.5 at dilution factor 10 (1.0 μg to 100 pg). Dilutions were brought to 0.5 mL with gel
phosphate as previously described and injected into 1 mouse per dilution. Mice wereperiodically monitored for up to 2 weeks post-injection.

In vitro assays
hiPSC-derived motor neurons purchased from Fujifilm Cellular Dynamics (Madison, WI,USA) were prepared as recommended by the manufacturer. rBoNT/X holotoxin (isolated
from E. coli) was pretreated with 10 mM DTT, then, toxin samples were treated withtrypsin or left untreated, and serial dilutions were applied to hiPSC-derived neuronalcell lysate cultures for 1 hour. VAMP2 cleavage was analyzed by western blot using
Synaptic Systems (Gottingen, Germany) Synaptobrevin2 antibody and goat anti-mouse
Ig secondary (Santa Cruz).
To test for toxin neutralization by anti-BoNT/X antiserum, trypsin-treated rBoNT/X
isolated from E. coli (20 nM) was incubated with anti-BoNT/X antiserum for 1 hour, andhiPSC-derived neurons were exposed to the antibody-BoNT/X mixture, BoNT/X alone,anti-BoNT/X antiserum alone, or culture media alone for 40 hours. Cell lysates wereanalyzed for VAMP2 cleavage via western blot, using syntaxin as a loading control.

Endopeptidase assay
Native BoNT/X derived from C. botulinum Strain 111 was assessed for in vitro cleav­age of VAMP2. Human GST-VAMP2 2-94 was prepared as described previously (47).
Briefly, cDNA from Hela cells was used as a template for the amplification of DNA
encoding the full length of VAMP2. The amplified DNAs were inserted into pGEX6P3
vector (Cytiva). To delete the transmembrane region mutagenesis, PCR was performed.
Recombinant proteins were expressed as C-terminally His-tagged and N-terminally
GST-tagged proteins in E. coli strain BL21-CodonPlus (DE3)-RIL (Agilent Technologies)
and purified using HisTrap HP (Cytiva) and GSTrap HP (Cytiva). BoNT/X was treatedwith reducing buffer (10 mM Tris-HCl pH 7.4, 20 mM NaCl, and 5 mM DTT) at 37°C
for 30 minutes, before being diluted to estimated final concentrations of 1, 5, 10, 20,
and 50 nM in cleavage buffer (10 mM Tris-HCl pH 7.4, 20 mM NaCl). BoNT/B1 wasdiluted to final concentrations of 5 and 50 nM. A control (0 nM) was treated as withall other samples but with no addition of toxin. Three-micromolar recombinant human
GST-VAMP2 2-94 was added to each toxin dilution and incubated at 37°C for 45 minutes.
Sample buffer was added, and samples were incubated at 95°C for 5 minutes before
analysis via western blot. In follow-up experiment, reduced native BoNT/X was pre-trea­
ted at 37°C for 30 minutes with 100 mM EDTA, 1 μg mouse anti-BoNT/X antiserum, and1 μg anti-BoNT/B rabbit polyclonal antibody or left untreated. Concentrations of BoNT/X
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Research Article(5 nM) and BoNT/B (100 nM) were selected to achieve approximately equal levels of
VAMP2 cleavage. Toxins were diluted in reducing buffer (10 mM Tris-HCl pH 7.4, 20 mM
NaCl, and 5 mM DTT).

hiPSC-derived neuronal cell assays
hiPSC-derived motor neurons purchased from Fujifilm Cellular Dynamics (Madison, WI,USA) were prepared as recommended by the manufacturer. Neurons were exposed toserial dilutions of the isolated trypsin-treated or untreated rBoNT/X for 24 hours (for toxin
expressed in E. coli) or 72 hours (for toxin expressed in C. botulinum) and analyzed for
VAMP2 cleavage by western blot.

Neuronal cell binding assay

In vivo mouse assays
Unless otherwise noted, all ICR mice for rBoNT/X analysis were purchased from Envigo(Indianapolis, IN), while ICR mice for native BoNT/X analysis were purchased from SLC(Hamamatsu, Japan).

Mouse bioassay
The mouse lethality bioassay is the current gold standard for BoNT detection in the
United States. Mice are injected i.p. with serial dilutions of toxin-containing samples
and observed for symptoms indicative of neuromuscular distress including ruffled fur,labored breathing and wasp waist, muscle weakness and paralysis, or death (48–50).
rBoNT/X expressed 24 hours in C. botulinum Hall A hyper tox- culture was sonicatedand solubilized, followed by either (i) collection of the extract supernatant (rBoNT/X
supernatant) or (ii) purification of the extract via size exclusion followed by ion exchange
chromatography and then pooling samples (rBoNT/X DEAE pool). Mice (n = 4) were
injected intraperitoneally with 500 µL of trypsin-treated or untreated rBoNT/X superna­tant (trypsin treatment 100 µg/mL, 30 minutes, 37°C) or rBoNT/X DEAE pool (trypsin
treatment 10 µg/mL, 30 minutes, 37°C) and observed for 4 days.
Four-week-old female ICR mice (n = 3) (SLC) were injected i.p. with 5 or 50 µg native
BoNT/X M-PTC in 300 µL 10 mM phosphate buffer (pH 6.0, 0.1% gelatin). Mice wereobserved for symptoms of motor-neuron impairment or other signs of distress at 6, 12,
24, 48, 72, 96, and 120 hours post-injection. As there were no mouse deaths associated
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Rat adrenal gland Phenochromocytoma (PC) 12 cells (provided by T. Kohda, Osaka
Metropolitan University, Osaka, Japan) were seeded onto 3.5-cm dish coated with
poly-L-lysine solution (Sigma-Aldrich) at a concentration of 3.0 × 105 cells per dish.
All culturing was performed at 37°C, 5% CO2. Cells were cultured 3 days (~70% confluent) before two successive PBS washes and addition of 0.5 mL ganglioside mixture(50 µg/mL in serum-free media) (Merck, Rahway, NJ). Cells were cultured for 24 hours andwashed twice with PBS, followed by exposure to BoNT/X or BoNT/B (estimated 300 nM
in 300 µL/well). After 4°C for 1 hour, the cells were washed three times with PBS to
remove toxin. Samples were fixed with 4% paraformaldehyde at room temperature for 30
minutes, followed by three washes with PBS. Samples were permeabilized and blockedwith 0.1% Triton- 1% bovine serum albumin (BSA, Sigma-Aldrich) at room temperaturefor 30 minutes. Add mouse polyclonal anti-BoNT/X antiserum and anti-BoNT/B rabbit
polyclonal antibodies and incubate at room temperature for 1 hour, followed by threewashes with PBS. Add Phalloidin-Alexa 568 (Thermo Fisher Scientific), Hoechst 33342(DOJINDO, Kumamoto, Japan), and either anti-mouse secondary (for BoNT/X sample) or
rabbit IgG-Alexa 488 (for BoNT/B sample). Repeat washing 3× with PBS, mount samples
using anti-fade kit, and image with a CSUX1 Confocal scanner unit (Yokogawa, Tokyo,
Japan) and IX71 Microscope (Olympus, Tokyo, Japan). The data were analyzed using
Metamorph software (Molecular Devices, San Jose, CA).

Research Article

mBiowith i.p. injections of recombinant or native BoNT/X, it was not possible to calculate a
median lethal dose (LD50) for these experiments.

DAS Assay
DAS assays were performed essentially as per K.R. Aoki (34), wherein mice were
injected in hindlimb gastrocnemius muscle using a 29 G needle (native BoNT/X; Terumo,
Somerset NJ). The opposite hindlimb was injected with an equal volume of vehicle or
10 mM phosphate buffer pH 6.0, 0.1% gelatin (native BoNT/X). Abduction in the mousehind digits was scored from DAS = 0 (normal) to DAS = 4 (maximal muscle weakness) at
indicated timepoints.
ACKNOWLEDGMENTS

AUTHOR AFFILIATIONS
1

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA
Department of Bacteriology, Kanazawa University, Kanazawa, Ishikawa, Japan3
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

2

AUTHOR ORCIDs
Brieana M. Gregg http://orcid.org/0009-0005-7142-8308
Takuhiro Matsumura http://orcid.org/0000-0002-3747-829X
Yukako Fujinaga http://orcid.org/0000-0001-8623-1376
Sabine Pellett http://orcid.org/0000-0001-8186-8810
FUNDING
FunderGrant(s)Author(s)CRDF Global (CRDF)

DAA3-20-66957-1,
DAA9-20-6659-1

Sabine Pellett

CRDF Global (CRDF)

20jk0210032h0001

Yukako Fujinaga

HHS | NIH | National Institute of Allergy
and Infectious Diseases (NIAID)

5R01AI139306-05

Sabine Pellett

Swedish Research Council

2022-03681

Pål Stenmark

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The authors wish to thank Dr. Nigel Minton for the use of the conjugal donor strain E.
coli CA434. Additionally, we thank the University of Wisconsin—Madison Select Agent
Program RO/AROs for their support in maintaining our Select Agent laboratory and the
UW—Madison ICDUR for DURC review of the manuscript.
The described study was supported by CRDF awards DAA3-20-66957-1,
DAA9-20-6659-1, and 20jk0210032h0001, with additional support from NIH:
5R01AI139396-05. This work was also supported by the Swedish Research Council(2022-03681) and the Swedish Cancer Society (20 1287 PjF) (P.S.).
B.G. wrote the manuscript, prepared the figures, optimized the expression of
rBoNT/X, and performed rBoNT/X cell-based cleavage assays. T.M. purified native
BoNT/X, performed in vitro and in situ BoNT/X activity, analyzed the potency in mice,and reviewed the manuscript. T.W. expressed, purified, and analyzed the BoNT/(X/A1)
chimera potency in mice and edited the manuscript. W.T. produced anti-BoNT/X
antibody for detection of recombinant and native BoNT/X, purified rBoNT/X, analyzed
the potency in mice, and edited the manuscript. M.B. performed cloning and expres­sion of rBoNT/X and reviewed the manuscript. P.S. provided the rBoNT/XRY expression
plasmids and developed the purification protocol. E.A.J conceptualized the design andacquired funding. Y.F. and S.P. contributed equally to the conceptual design, acquired
funding, supervised the experiments, and prepared the manuscript.

mBio

Research Article

FunderGrant(s)Author(s)Swedish Cancer Foundation

20 1287 PjF

Pål Stenmark

University of Wisconsin- Madison Food
Research Institute

Sabine Pellett

AUTHOR CONTRIBUTIONS
Brieana M. Gregg, Conceptualization, Data curation, Formal analysis, Investigation,
Methodology, Writing – original draft, Writing – review and editing | Takuhiro Matsu­
mura, Conceptualization, Data curation, Formal analysis, Investigation, Methodology,
Writing – review and editing | Travis G. Wentz, Data curation, Formal analysis, Inves­tigation, Methodology, Writing – review and editing | William H. Tepp, Conceptualiza­
tion, Data curation, Formal analysis, Investigation, Methodology, Writing – review andediting | Marite Bradshaw, Conceptualization, Data curation, Formal analysis, Investiga­
tion, Methodology, Supervision, Writing – review and editing | Pål Stenmark, Resources,
Writing – review and editing | Eric A. Johnson, Conceptualization, Funding acquisition,
Writing – review and editing | Yukako Fujinaga, Conceptualization, Funding acquisition,
Investigation, Methodology, Project administration, Resources, Supervision, Writing –review and editing | Sabine Pellett, Conceptualization, Funding acquisition, Investiga­
tion, Methodology, Project administration, Resources, Supervision, Writing – review andediting
ADDITIONAL FILES
The following material is available online.
Supplemental Material
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