Progenitor Toxin Research Articles

NTNH protein: more than a bodyguard for botulinum neurotoxins.

As one of the most fatal substances, botulinum neurotoxins (BoNTs) have never acted solo to accomplish their formidable missions. Most notably, nontoxic nonhemagglutinin (NTNH), a protein co-secreted with BoNT by bacteria, plays critical roles to stabilize and protect BoNT by tightly associating with it to form the minimal progenitor toxin complex (M-PTC). A new cryo-EM structure of the M-PTC of a BoNT-like toxin from Weissella oryzae (BoNT/Wo) reveals similar assembly modes between M-PTC/Wo and that of other BoNTs, yetalso reveals some unique structural features of NTNH/Wo. These findings shed new light on the potential versatile roles of NTNH during BoNT intoxication.

Complete subunit structure of serotype C and D botulinum progenitor toxin complex induces vacuolation in the specific epithelial cell line

Clostridium botulinum produces seven botulinum neurotoxin (BoNT) serotypes. In nature, BoNT exists as a part of the progenitor toxin complex (PTC) through associations with neurotoxin associated proteins (NAPs), including nontoxic nonhemagglutinin and hemagglutinin (HA) complex, consists of HA-70, HA-17 and HA-33. Because PTC displays higher oral toxicity than pure BoNTs, NAPs play a critical role in food poisoning. In a previous study, we demonstrated that the NAP complex in mature large-sized PTC (L-PTC) from serotypes C and D concomitantly induced cell death and cytoplasmic vacuolation in the rat intestinal epithelial cell line IEC-6. Here, we found that the serotype D NAP complex induces only cytoplasmic vacuolation in the normal rat kidney cell line NRK-52E without reducing cell viability. NAP complexes from serotype A and B L-PTCs did not affect cell viability or cytoplasmic vacuolation in IEC-6 and NRK-52E cells. Furthermore, we assessed the effect of immature L-PTCs with fewer HA-33/HA-17 trimers (two HA-33 and one HA-17) than mature L-PTCs on cell viability and cytoplasmic vacuolation in IEC-6 and NRK-52E cells. As a result, mature L-PTCs with the maximum number of HA-33/HA-17 trimers displayed the greatest potency. Consequently, the reduction in cell viability and vacuolation induction are related to the number of HA-33/HA-17 trimers in PTC. The discovery of an epithelial cell model where botulinum PTC specifically induces vacuolization may help clarify the unknown cytotoxicity of PTC, which plays an important role in the trans-epithelial transport of the toxin.

Botulinum Hemagglutinin: Critical Protein for Adhesion and Absorption of Neurotoxin Complex in Host Intestine.

Botulinum hemagglutinin (HA) is one of the auxiliary protein components of the botulinum neurotoxin (BoNT) complex, the most lethal toxin known. HA promotes the intestinal absorption of BoNT by at least two mechanisms, resulting in high oral toxicity. One of the mechanisms is the attachment of large progenitor toxin complexes (L-PTCs) to the cell surface of the intestinal epithelium by the carbohydrate-binding activity of HA. The other is epithelial barrier disruption by the E-cadherin-binding activity of HA. The carbohydrate-binding activity of HA also promotes attachment to the basolateral cell surface, which increases the frequency of contact between HA and E-cadherin. Together, the carbohydrate-binding activity of HA is critical for the intestinal absorption of BoNTs. The trimeric triskelion-shaped structure of HA confers the multivalent binding to its ligands and increases the pathogenic biological activities of HA.

The role of human serum albumin and neurotoxin associated proteins in the formulation of BoNT/A products

Botulinum neurotoxin (BoNT) is synthesized as a progenitor toxin complex (PTC) by Clostridium botulinum. This PTC comprises, in addition to the neurotoxin itself, neurotoxin associated proteins (NAPs) which are composed of three hemagglutinins and one non-toxic, non-hemagglutinin protein. After oral ingestion, these NAPs protect the neurotoxin from the low pH and proteases in the gastrointestinal tract and play a role in the entry via the intestinal barrier. Two of the three therapeutically used botulinum neurotoxin serotype A (BoNT/A) products (onabotulinumtoxinA and abobotulinumtoxinA) contain different amounts of NAPs, while incobotulinumtoxinA, lacks these proteins. In addition, human serum albumin (HSA) that is supposed to stabilize BoNT/A is added at different concentrations. Up to now, the function of the NAPs and HSA after parenteral therapeutic application is not completely understood.To investigate the influence of NAPs and HSA on potency of BoNT/A, we used the ex vivo mouse phrenic nerve hemidiaphragm assay.Increasing doses of HSA resulted dose-dependently in a more pronounced effect of BoNT/A. Though, a plateau was reached with concentrations of 0.8 mg/ml HSA and higher, the accessory addition of NAPs in a relevant amount (4 ng/ml) did not further enhance the effect of BoNT/A.In conclusion, in our ex vivo assay an adequate concentration of HSA prevented BoNT/A from loss of effect and supplementary NAPs did not alter this effect. A confirmation of these data in an in vivo assay is still lacking. However, it might be supposed that even in clinically applied BoNT/A products an increase of HSA accompanied by the avoidance of NAPs could potentially reduce the injected dose and, thus, the risk of unwanted side effects, the treatment costs as well as the risk of a secondary therapy failure due to BoNT/A neutralizing antibodies.

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.

The Structure and Classification of Botulinum Toxins.

Botulinum neurotoxins (BoNTs) are a family of bacterial protein toxins produced by various Clostridium species. They are traditionally classified into seven major serotypes (BoNT/A-G). Recent progress in sequencing microbial genomes has led to an ever-growing number of subtypes, chimeric toxins, BoNT-like toxins, and remotely related BoNT homologs, constituting an expanding BoNT superfamily. Recent structural studies of BoNTs, BoNT progenitor toxin complexes, tetanus neurotoxin (TeNT), toxin-receptor complexes, and toxin-substrate complexes have provided mechanistic understandings of toxin functions and the molecular basis for their variations. The growing BoNT superfamily of toxins present a natural repertoire that can be explored to develop novel therapeutic toxins, and the structural understanding of their variations provides a knowledge basis for engineering toxins to improve therapeutic efficacy and expand their clinical applications.

Botulinum and Tetanus Neurotoxins.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Study of response of Swiss Webster mice to light subunit of mushroom tyrosinase

The light subunit of mushroom, Agaricus bisporus, tyrosinase (LSMT), has been identified as an extrinsic component of the enzyme. Its function is unknown, but it can cross an epithelial cell layer, which suggests that it can be absorbed by the intestine. A similar capability has been demonstrated for the HA-33 component of the progenitor toxin from Clostridium botulinum, which is the closest structural homolog of LSMT. Unlike HA-33, LSMT appears to be non-immunogenic as shown by preliminary tests in Swiss Webster mice. We investigated the immunogenicity and histopathology of LSMT in mice to determine its safety in vivo. LSMT did not evoke generation of antibodies after prolonged periods of intraperitoneal administration. Histopathological observations confirmed the absence of responses in organs after twelve weekly administrations of LSMT. We found that LSMT is not toxic and is less immunogenic than the C. botulinum HA-33 protein, which supports further research and development for pharmaceutical application.

Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins.

Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs) to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH), and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC), but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs) which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex.

Dynamics of the Large Progenitor Toxin Complex of Clostridium botulinum

Botulinum neurotoxins (BoNTs) are one of the potent toxins in nature but the toxicity is immediately eliminated by the harsh environment in the digestive tract because BoNTs are just a protein. However, BoNTs can get to synaptic junctions thanks to support of other proteins by aggregating with each other, combining with BoNTs and forming a large progenitor toxin complex (L-PTC). In order to explain how the complex formation enables the BoNTs to intrude into our body, we found that the three-dimensional structure of the L-PTC consists of an ovoid body with three legs and speculated important roles in the body and the legs. In the legs part, it is helpful for promoting absorption especially from the small intestine. Because experimental results showed that the legs are flexible and have specific binding sites of saccharides, the flexibilities may help the L-PTC to easily access the binding sites to the saccharides on the intestinal surface. However, such flexibilities have been only investigated by experimental methods. This means that we still have not objectively discussed what motions are generated from the shape of the L-PTC and how the structure is changed gradually. Therefore, we developed a new method integrating an analysis based on anisotropic network model and principal component analyses in order to measure the dynamics of the large-sized protein consisting of several subunits. The results showed that the L-PTC had characteristic motions which have large movements of the three legs. In addition, the flexible motions were appeared regardless of the theoretical models. Our application to measure the dynamics of the L- PTC suggested the importance of the flexibility which enables the L-PTC to break the epithelial barrier. We hope that the activity of the L-PTC is applied for developing a new oral drug delivery system.

Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.

BackgroundThe disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found.MethodsComponents of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS.ResultsGel bands were identified with sequence coverages of 91 % for BoNT/G, 91 % for NTNH, 89 % for HA-70, and 88 % for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93 % sequence coverage.ConclusionsThe BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.

The long journey of botulinum neurotoxins into the synapse

Botulinum neurotoxins (BoNT) cause the disease botulism, a flaccid paralysis of the muscle. They are also very effective, widely used medicines applied locally in sub-nanogram quantities. BoNTs are released together with several non-toxic, associated proteins as progenitor toxin complexes (PCT) by Clostridium botulinum to become highly potent oral poisons ingested via contaminated food. They block the neurotransmission in susceptible animals and humans already in nanogram quantities due to their specific ability to enter motoneurons and to cleave only selected neuronal proteins involved in neuroexocytosis.BoNTs have developed a sophisticated strategy to passage the gastrointestinal tract and to be absorbed in the intestine of the host to finally attack neurons. A non-toxic non-hemagglutinin (NTNHA) forms a binary complex with BoNT to protect it from gastrointestinal degradation. This binary M-PTC is one component of the bi-modular 14-subunit ∼760 kDa large progenitor toxin complex. The other component is the structurally and functionally independent dodecameric hemagglutinin (HA) complex which facilitates the absorption on the intestinal epithelium by glycan binding. Subsequent to its transcytosis the HA complex disrupts the tight junction of the intestinal barrier from the basolateral side by binding to E–cadherin. Now, the L-PTC can also enter the circulation by paracellular routes in much larger quantities. From here, the dissociated BoNTs reach the neuromuscular junction and accumulate via interaction with polysialo gangliosides, complex glycolipids, on motoneurons at the neuromuscular junction. Subsequently, additional specific binding to luminal segments of synaptic vesicles proteins like SV2 and synaptotagmin leads to their uptake. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had exploited before to enter their target cells, via specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which constitute the core components of the cellular membrane fusion machinery.

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.

Structural Basis of the pH-Dependent Assembly of a Botulinum Neurotoxin Complex

Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.

High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex

Botulinum neurotoxins (BoNTs) are produced as progenitor toxin complexes (PTCs) by Clostridium botulinum. The PTCs are composed of BoNT and non-toxic neurotoxin-associated proteins (NAPs), which serve to protect and deliver BoNT through the gastrointestinal tract in food borne botulism. HA33 is a key NAP component that specifically recognizes host carbohydrates and helps enrich PTC on the intestinal lumen preceding its transport across the epithelial barriers. Here, we report the crystal structure of HA33 of type B PTC (HA33/B) in complex with lactose at 1.46Å resolution. The structural comparisons among HA33 of serotypes A–D reveal two different HA33–glycan interaction modes. The glycan-binding pockets on HA33/A and B are more suitable to recognize galactose-containing glycans in comparison to the equivalent sites on HA33/C and D. On the contrary, HA33/C and D could potentially recognize Neu5Ac as an independent receptor, whereas HA33/A and B do not. These findings indicate that the different oral toxicity and host susceptibility observed among different BoNT serotypes could be partly determined by the serotype-specific interaction between HA33 and host carbohydrate receptors. Furthermore, we have identified a key structural water molecule that mediates the HA33/B–lactose interactions. It provides the structural basis for development of new receptor-mimicking compounds, which have enhanced binding affinity with HA33 through their water-displacing moiety.

Botulinum Neurotoxin A Complex Recognizes Host Carbohydrates through Its Hemagglutinin Component

Botulinum neurotoxins (BoNTs) are potent bacterial toxins. The high oral toxicity of BoNTs is largely attributed to the progenitor toxin complex (PTC), which is assembled from BoNT and nontoxic neurotoxin-associated proteins (NAPs) that are produced together with BoNT in bacteria. Here, we performed ex vivo studies to examine binding of the highly homogeneous recombinant NAPs to mouse small intestine. We also carried out the first comprehensive glycan array screening with the hemagglutinin (HA) component of NAPs. Our data confirmed that intestinal binding of the PTC is partly mediated by the HA moiety through multivalent interactions between HA and host carbohydrates. The specific HA-carbohydrate recognition could be inhibited by receptor-mimicking saccharides.

Insilico Studies on HA 33 Subunit of Haemagglutinin of Clostridium botulinum Construction of Three Dimensional Model and Validation

Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. The Clostridia releases these as protein aggregate, termed as the progenitor toxins. Heamagglutin – 33 (HA-33) plays an essential role in the agglutination activity of the botulinum progenitor toxin; it assists in binding and translocating the progenitor toxins through the alimentary epithelial barriers. A three-dimensional homology model of HA-33 is demonstrated in this paper based upon the crystal structure of HA1 subcomponent of botulinum type C progenitor toxin complexed with galactose. The three dimensional model was designed using MODELLER (9v11) a comparative modelling tool that built a model based on an alignment of a sequence of HA-33 based on the known structure of botulinum type C progenitor toxin complexed with N-acetylgalactosamine. The modelled structure was then evaluated using SAVES. The structural dynamics of the modelled protein was performed by elNemo and based on the scores obtained, it was found to be a reliable and accurate three dimensional structure of the Hemeagglutin component. It gives an idea about the three dimensional structure of a protein which will would serve as a guide to a detailed understanding of its participation in the process of agglutination. This could also aid in molecular drug designing against botulism.

Comparison of the immunogenicity of botulinum toxin type A and the efficacy of A1 and A2 neurotoxins in animals with A1 toxin antibodies

One issue with botulinum toxin type A products is a reduced therapeutic response in patients that have been injected with frequent dosing over a prolonged period. A possible cause of this is hemagglutinin, found in progenitor toxins, displaying adjuvant activity, enhancing antibody production against the toxin. We investigated whether there is any difference in immunogenicity between the LL toxin-derived subtype A1 (A1LL) and the neurotoxin-derived subtypes A1 and A2 (A1NTX and A2NTX, respectively), and investigated whether A2NTX is effective in animals which produce antibodies against A1LL.Neutralizing antibodies were detected in the A1LL-administered group; however, they were not detected in swine and rabbits administered multiple doses of A2NTX. These results indicate that A2NTX has a lower immunogenicity than A1LL. In rats with neutralizing antibodies, produced by the administration of A1LL, that were administered either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. In human sera immunized with the botulinum toxoid vaccine (containing LL, L, and M toxoid derived subtype A1) reacted with either A1NTX or A2NTX, A2NTX showed more potent inhibitory neuromuscular transmission than A1NTX. This suggests that A2NTX has a greater therapeutic value in humans who have neutralizing antibodies against the A1 toxin.

Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.

Analysis of the Mechanisms That Underlie Absorption of Botulinum Toxin by the Inhalation Route

Botulinum toxin is a highly potent oral and inhalation poison, which means that the toxin must have an efficient mechanism for penetration of epithelial barriers. To date, three models for toxin passage across epithelial barriers have been proposed: (i) the toxin itself undergoes binding and transcytosis; (ii) an auxiliary protein, HA35, transports toxin from the apical to the basal side of epithelial cells; and (iii) an auxiliary protein, HA35, acts on the basal side of epithelial cells to disrupt tight junctions, and this permits paracellular flux of toxin. These models were evaluated by studying toxin absorption following inhalation exposure in mice. Three types of experiments were conducted. In the first, the potency of pure neurotoxin was compared with that of progenitor toxin complex, which contains HA35. The results showed that the rate and extent of toxin absorption, as well as the potency of absorbed toxin, did not depend upon, nor were they enhanced by, the presence of HA35. In the second type of experiment, the potencies of pure neurotoxin and progenitor toxin complex were compared in the absence or presence of antibodies on the apical side of epithelial cells. Antibodies directed against the neurotoxin protected against challenge, but antibodies against HA35 did not. In the final type of experiment, the potency of pure neurotoxin and toxin complex was compared in animals pretreated to deliver antibodies to the basal side of epithelial cells. Once again, antibodies directed against the neurotoxin provided resistance to challenge, but antibodies directed against HA35 did not. Taken collectively, the data indicate that the toxin by itself is capable of crossing epithelial barriers. The data do not support any hypothesis in which HA35 is essential for toxin penetration of epithelial barriers.