Molecular engineering of a minimal E-cadherin inhibitor protein derived from Clostridium botulinum hemagglutinin.

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Neutralization mechanism of human monoclonal antibodies against type B botulinum neurotoxin.

Botulism is a deadly neuroparalytic condition caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum and related species. Toxin-neutralizing antibodies are the most effective treatments for BoNT intoxication. We generated human monoclonal antibodies neutralizing type B botulinum neurotoxin (BoNT/B), designated M2 and M4. The combination of these antibodies exhibited a strong neutralizing effect against BoNT/B toxicity. In this study, we analyzed the mechanisms of action of these antibodies in vitro. M4 binds to the C-terminus of the heavy chain (the receptor-binding domain) and inhibits BoNT/B binding to neuronal PC12 cells. Although M2 recognized the light (L) chain (the metalloprotease domain), it did not inhibit substrate (VAMP2) cleavage in the cleavage assay. M2 increased the surface localization of BoNT/B in PC12 cells at a later time point, suggesting that M2 inhibits the translocation of the L chain from synaptic vesicles to the cytosol. These results indicate that M2 and M4 inhibit the different processes of BoNT/B individually and that multistep inhibition is important for the synergistic effect of the combination of monoclonal antibodies. Our findings may facilitate the development of effective therapeutic antibodies against BoNTs.

Adult-onset botulism in a Japanese woman with prolonged spore excretion.

We report a case of an 80-year-old woman with botulism from 2020 in Osaka, Japan. The patient complained of dysarthria and dizziness. On the same day, the patient developed respiratory failure, and was intubated and placed on mechanical ventilation. Subsequently, ophthalmoparesis and quadriparesis progressed rapidly. Ten days after onset, the patient failed to respond to any external stimulation. Blood tests showed anemia, and computed tomography revealed undiagnosed cervical cancer. Initially, diagnosis of neuromuscular junction disorder and acute motor neuropathy, including paraneoplastic syndrome, were considered. However, intravenous immunoglobulin therapy and plasma exchange were ineffective. A fecal sample on day 30 showed a large number of C. botulinum spores. On day 34, a mouse bioassay revealed botulinum toxin type A in the patient's serum; therefore, a botulinum antitoxin was administered. Later, the patient's muscle strength was gradually improved. However, severe muscle paralysis persisted, and the patient died of cachexia owing to cervical cancer on day 196. The etiology of this case was unknown because no contaminated food was identified during an inspection of the patient's home. Fecal 16S rRNA gene sequencing revealed dysbiosis of the intestinal microbiota with abundant Enterococcus species. Long-lasting excretion of substantial botulinum spores even on day 30 indicated colonization of C. botulinum in the intestinal tract. This case suggests that C. botulinum colonization with co-existing intestinal dysbiosis may be associated with severe and prolonged symptoms of botulism.

Rare toxin A-negative and toxin B-positive strain of Clostridioides difficile from Japan lacking a complete tcdA gene.

INTRODUCTION: Clostridioides difficile (C. difficile) produces three kinds of toxins: toxin A (enterotoxin), toxin B (cytotoxin), and C. difficile transferase (CDT), a binary toxin. Some strains show positivity only for toxin B. These strains reportedly possess a gene for toxin A, tcdA. However, toxin A production is inhibited due to a mutated stop codon and/or deletion within the tcdA gene. Here for the first case in Japan, we describe toxin genomes and proteins of a strain possessing only toxin B and lacking a complete tcdA gene, along with clinical manifestations. METHODS: C. difficile was isolated from the bloody stool of a 60-year-old female patient treated with meropenem. Although a rapid detection kit of toxins (C. DIFF QUIK CHEK COMPLETE®, TechLab, Blacksburg, VA, USA) showed positivity, Western blotting detected no toxins. Therefore, we explored the strain's toxin genes and their sequences to determine whether the strain possessed a toxin. RESULTS: Polymerase chain reaction did not identify toxin genes. Whole-genome sequencing analysis showed that a gene for toxin A, tcdA, was completely deleted in the strain. Moreover, 701 mutations and some deletions/insertions were identified on the tcdB gene. CONCLUSIONS: We isolated a rare strain of C. difficile producing only toxin B and lacking a complete tcdA gene herein Japan. The possibility of a false negative needs to be considered with a genetic method for a diagnose of C. difficile infection.

Effects of antibiotics on the viability of and toxin production by Clostridium botulinum.

Clostridium botulinum causes infant and adult intestinal botulism by colonizing in the intestine and producing botulinum neurotoxin (BoNT). Antimicrobial agents are not currently used for treatment due to the potential facilitation of BoNT production and bacterial cell lysis, which releases toxins into the intestinal lumen. In this study, we analyzed effects of four antibiotics on the viability of and BoNT production by four C. botulinum group I strains. Our results indicate that metronidazole rapidly reduced their viability without enhancing BoNT production. Antibiotics with these properties may promote elimination of C. botulinum from the intestines while maintaining low levels of BoNT.

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity.

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of ß-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

Uptake of botulinum neurotoxin in the intestine.

Foodborne and intestinal botulism are the most common forms of human botulism; both result from the absorption of botulinum neurotoxin (BoNT) from the digestive tract into the circulation. BoNT is a large protein toxin (approximately 150 kDa), but it is able to pass through the epithelial barrier in the digestive tract. Recent cellular and molecular biology studies have begun to unravel the mechanisms by which this large protein toxin crosses the intestinal epithelial barrier. This review provides an overview of current knowledge relating to the absorption of botulinum toxins (BoNT and BoNT complex) from the gastrointestinal tract, with particular emphasis on the interaction of these toxins with the intestinal epithelial barrier.

Botulinum neurotoxin X lacks potency in mice and in human neurons.

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 disulfide-bonded dichain polypeptide similar to other BoNTs. Both the purified native and the recombinant 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. Intraperitoneal 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 neuronal cell 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 a potent 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 more distantly related homologs are being identified. The discovery of BoNT/X via bioinformatic 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 time assessed both recombinantly produced and native purified BoNT/X for its vertebrate neurotoxicity.

Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88/TRIF Signaling in vitro.

Clostridium botulinum produces botulinum neurotoxin complexes that cause botulism. Previous studies elucidated the molecular pathogenesis of botulinum neurotoxin complexes; however, it currently remains unclear whether other components of the bacterium affect host cells. Recent studies provided insights into the role of bacterial membrane vesicles (MVs) produced by some bacterial species in host immunity and pathology. We herein examined and compared the cellular effects of MVs isolated from four strains of C. botulinum with those of closely related Clostridium sporogenes and two strains of the symbiont Clostridium scindens. MVs derived from all strains induced inflammatory cytokine expression in intestinal epithelial and macrophage cell lines. Cytokine expression was dependent on myeloid differentiation primary response (MyD) 88 and TIR-domain-containing adapter-inducing interferon-ß (TRIF), essential adaptors for toll-like receptors (TLRs), and TLR1/2/4. The inhibition of actin polymerization impeded the uptake of MVs in RAW264.7 cells, however, did not reduce the induction of cytokine expression. On the other hand, the inhibition of dynamin or phosphatidylinositol-3 kinase (PI3K) suppressed the induction of cytokine expression by MVs, suggesting the importance of these factors downstream of TLR signaling. MVs also induced expression of Reg3 family antimicrobial peptides via MyD88/TRIF signaling in primary cultured mouse small intestinal epithelial cells (IECs). The present results indicate that MVs from C. botulinum and related clostridial species induce host innate immune responses.

Crystallization and preliminary crystallographic studies of the HA3 subcomponent of the type B botulinum neurotoxin complex.

The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values. Form I belonged to space group R32, with unit-cell parameters a = b = 357.4, c = 249.5 Å, α = ß = 90, γ = 120°. Form II belonged to space group I4(1)32, with unit-cell parameters a = b = c = 259.0 Å, α = ß = γ = 90°. Diffraction data were collected from these crystals to a resolution of 3.0 Å for both form I and form II.

Functional Analysis of Botulinum Hemagglutinin (HA).

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most potent toxin and produced as a complex with non-toxic components. Food-borne botulism is caused by the ingestion of these BoNT complexes. Hemagglutinin (HA), one of the non-toxic components, is known to have lectin (carbohydrate binding) activity and E-cadherin-binding activity. These activities promote the intestinal absorption of BoNT. To elucidate the mechanism of the onset of food-borne botulism, we focused on the role of HA in the intestinal absorption of BoNT. We describe the functional analysis methods for HA, including the expression of recombinant proteins, binding to glycoproteins and epithelial cells, and localization in mouse intestinal tissue.

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B.

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

Collagen adhesion gene is associated with bloodstream infections caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption.

The type B botulinum neurotoxin (BoNT) elicits flaccid paralysis and death in humans by intoxicating peripheral nerves after oral absorption. Here, we examine the function of the haemagglutinin (HA), a non-toxic component of the large 16S BoNT complex. We find that the HA acts in the intestine to disrupt epithelial barrier function by opening intercellular tight and adherens junctions. This allows transport of BoNT and other large solutes into the systemic circulation and explains how the type B BoNT complexes are efficiently absorbed. In vitro, HA appears to act on the epithelial cell via the basolateral membrane only, suggesting the possibility of another step in the absorptive process. These studies show that the 16S BoNT complex is a multifunctional protein assembly equipped with the machinery to efficiently breach the intestinal barrier and act systemically on peripheral nerves.

[Mechanism of intestinal absorption of botulinum neurotoxin complex].

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum and related species cause botulism, a neuroparalytic disease associated with a high mortality. BoNTs are always produced as large protein complexes (progenitor toxin complexes, PTCs) through association with non-toxic components (NAPs) including hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNHA). Food-borne botulism is caused by the ingestion of PTCs. PTCs in the gastrointestinal tract cross the intestinal epithelial barrier, enter the blood stream, and reach the nerve endings, where BoNTs cleave the SNAREs required for vesicle fusion. Consequently, BoNTs inhibit neurotransmitter release and cause paralysis. To cause food-borne botulism, BoNTs must traverse the intestinal epithelial barrier. However, the mechanism used to cross this barrier remains unclear. Using an in vitro epithelial barrier system, we previously showed that the interaction of HA with E-cadherin results in disruption of tight junctions. Furthermore, we previously reported that microfold (M) cells in the follicle-associated epithelium (FAE) of mouse Peyer's patches (PPs) are major sites where type A1 BoNT breaches the intestinal epithelial barrier. Here, I would like to demonstrate an ingenious invasion mechanism of the BoNT complex.

Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.

Botulinum neurotoxin is conventionally divided into seven serotypes, designated A-G, and is produced as large protein complexes through associations with non-toxic components, such as hemagglutinin (HA) and non-toxic non-HA. These non-toxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier-disrupting activity. Type A and B HAs disrupt E-cadherin-mediated cell adhesion, and, in turn, the intercellular epithelial barrier. Type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pre-treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to a-series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked expression of ST-I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST-I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a-series gangliosides, suggesting a GM3-specific mechanism. Our results demonstrate that HA/C affects cells in a GM3-dependent manner.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

Functional dissection of the Clostridium botulinum type B hemagglutinin complex: identification of the carbohydrate and E-cadherin binding sites.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin.

Botulinum neurotoxin is produced by Clostridium botulinum and forms large protein complexes through associations with nontoxic components. We recently found that hemagglutinin (HA), one of the nontoxic components, disrupts the intercellular epithelial barrier; however, the mechanism underlying this phenomenon is not known. In this study, we identified epithelial cadherin (E-cadherin) as a target molecule for HA. HA directly binds E-cadherin and disrupts E-cadherin-mediated cell to cell adhesion. Although HA binds human, bovine, and mouse E-cadherin, it does not bind rat or chicken E-cadherin homologues. HA does not interact with other members of the classical cadherin family such as neural and vascular endothelial cadherin. Expression of rat E-cadherin but not mouse rescues Madin-Darby canine kidney cells from HA-induced tight junction (TJ) disruptions. These data demonstrate that botulinum HA directly binds E-cadherin and disrupts E-cadherin-mediated cell to cell adhesion in a species-specific manner and that the HA-E-cadherin interaction is essential for the disruption of TJ function.