Clostridial Neurotoxins Research Articles

Closed Genome Sequence of Chryseobacterium piperi Strain CTMT/ATCC BAA-1782, a Gram-Negative Bacterium with Clostridial Neurotoxin-Like Coding Sequences.

ABSTRACTClostridial neurotoxins, including botulinum and tetanus neurotoxins, are among the deadliest known bacterial toxins. Until recently, the horizontal mobility of this toxin gene family appeared to be limited to the genus Clostridium. We report here the closed genome sequence of Chryseobacterium piperi, a Gram-negative bacterium containing coding sequences with homology to clostridial neurotoxin family proteins.

Hsp90 and Thioredoxin-Thioredoxin Reductase enable the catalytic activity of Clostridial neurotoxins inside nerve terminals

Botulinum (BoNTs) and tetanus (TeNT) neurotoxins are the most toxic substances known and form the growing family of Clostridial neurotoxins (CNT), the etiologic agents of botulism and tetanus. CNT are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol, where its substrates, the three SNARE proteins, are localized. L translocation is accompanied by unfolding and, once delivered on the cytosolic side of the endosome membrane, it has to be reduced and reacquire the native fold to be active. The Thioredoxin-Thioredoxin Reductase system (Trx-TrxR) specifically reduces the interchain disulfide bond while the cytosolic chaperone protein Hsp90 mediates L refolding. Both steps are essential for CNT activity and their inhibition efficiently blocks the neurotoxicity in cultured neurons and mice. Trx and its reductase physically interact with Hsp90 and are loosely bound to the cytosolic side of synaptic vesicles, the organelle exploited by CNT to enter nerve terminals and wherefrom L is translocated into the cytosol. Therefore, Trx, TrxR and Hsp90 orchestrate a chaperone-redox molecular machinery that enables the catalytic activity of the L inside nerve terminals. Given the fundamental role of L reduction and refolding, this machinery represents a rational target for the development of mechanism-based antitoxins.

The travel diaries of tetanus and botulinum neurotoxins

Tetanus (TeNT) and botulinum (BoNT) neurotoxins, the causative agents of tetanus and botulism, respectively, are the most potent toxic molecules known to mankind. This extreme potency is attributed to: i) their specificity for essential components of the neurotransmitter release machinery present at vertebrate synapses, and ii) their high-affinity targeting to motor neurons by binding to polysialogangliosides and protein receptors. Comprising the clostridial neurotoxin family, TeNT and BoNTs engage distinct surface receptors and intracellular sorting pathways in neurons. BoNTs bind to the intraluminal domain of specific synaptic vesicle proteins that are exposed to the extracellular milieu upon exocytosis, and are taken up by synaptic vesicle recycling. A sizeable proportion of BoNT molecules remain at the neuromuscular junction, where their protease moiety is released into the cytoplasm, blocking synaptic transmission and causing flaccid paralysis. In contrast, TeNT undergoes binding to specific components of the basal membrane at the neuromuscular junction, is endocytosed into motor neurons and sorted to axonal signalling endosomes. Following this, TeNT is transported to the soma of motor neurons located in the spinal cord or brainstem, and then transcytosed to inhibitory interneurons, where it blocks synaptic transmission. TeNT-induced impairment of inhibitory input leads to hyperactivity of motor neurons, causing spastic paralysis, which is the hallmark of tetanus. This review examines the molecular mechanisms leading to the entry, sorting and intracellular trafficking of TeNT and BoNTs.

The structure of the tetanus toxin reveals pH-mediated domain dynamics.

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a "closed" domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.

Insights into the Mechanisms by Which Clostridial Neurotoxins Discriminate between Gangliosides.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the causative agents of the paralytic diseases botulism and tetanus, respectively. Entry of toxins into neurons is mediated through initial interactions with gangliosides, followed by binding to a protein co-receptor. Herein, we aimed to understand the mechanism through which individual neurotoxins recognize the carbohydrate motif of gangliosides. Using cell-based and in vitro binding assays, in conjunction with structure-driven site-directed mutagenesis, a conserved hydrophobic residue within the BoNTs that contributes to both affinity and specificity toward Sia5-containing gangliosides was identified. We demonstrate that targeted mutations within the Sia5 binding pocket result in the generation of neurotoxins that either bind and enter cells more efficiently (BoNT/A1 and BoNT/B) or display altered ganglioside binding specificity (TeNT). These data support a model in which recognition of Sia5 is largely driven by hydrophobic interactions between the sugar and the Sia5 binding site.

Mechanism of inhibition of botulinum neurotoxin type A light chain by two quinolinol compounds

Quinolinol-based compounds are a promising starting point for discovery of effective inhibitors of the clostridial neurotoxin, botulinum neurotoxin type A light chain (BoNT/A LC). Insights into the mechanism of inhibition by quinolinol compounds facilitate interpretation of docking data and inhibitor optimization. In this study, a fluorogenic substrate of BoNT/A, SNAPtide, was used to study the mechanism by which two new quinolinol compounds, MSU58 and MSU84, with IC50 values of 3.3 μM and 5.8 μM, respectively, inhibit BoNT/A LC. Kinetic studies and model discrimination analysis showed both compounds to be competitive inhibitors of BoNT/A LC with inhibition constants (KI) 3.2 μM and 6.2 μM for MSU58 and MSU84, respectively. These data indicate that the inhibitors bind in the BoNT/A LC active site and that inhibitor binding is mutually exclusive with the binding of the substrate. This is the first study to report the competitive inhibition of BoNT/A LC by quinolinol compounds. These data help define the inhibitor binding pocket and, along with structure activity relationship studies, provide immediate direction for further compound synthesis.

Uptake of Clostridial Neurotoxins into Cells and Dissemination.

Clostridial neurotoxins, botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT), are potent toxins, which are responsible for severe neurological diseases in man and animals. BoNTs induce a flaccid paralysis (botulism) by inhibiting acetylcholine release at the neuromuscular junctions, whereas TeNT causes a spastic paralysis (tetanus) by blocking the neurotransmitter release (glycine, GABA) in inhibitory interneurons within the central nervous system. Clostridial neurotoxins recognize specific receptor(s) on the target neuronal cells and enter via a receptor-mediated endocytosis. They transit through an acidic compartment which allows the translocation of the catalytic chain into the cytosol, a prerequisite step for the intracellular activity of the neurotoxins. TeNT migrates to the central nervous system by using a motor neuron as transport cell. TeNT enters a neutral pH compartment and undergoes a retrograde axonal transport to the spinal cord or brain, where the whole undissociated toxin is delivered and interacts with target neurons. Botulism most often results from ingestion of food contaminated with BoNT. Thus, BoNT passes through the intestinal epithelial barrier mainly via a transcytotic mechanism and then diffuses or is transported to the neuromuscular junctions by the lymph or blood circulation. Indeed, clostridial neurotoxins are specific neurotoxins which transit through a transport cell to gain access to the target neuron, and use distinct trafficking pathways in both cell types.

Hsp90, thioredoxin, and thioredoxin reductase form a chaperone-redox machinery enabling the catalytic activity of clostridial neurotoxins inside nerve terminals

Hsp90, thioredoxin, and thioredoxin reductase form a chaperone-redox machinery enabling the catalytic activity of clostridial neurotoxins inside nerve terminals

Structural studies of clostridial neurotoxins binding to ganglioside receptors

Structural studies of clostridial neurotoxins binding to ganglioside receptors

Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals.

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised. L translocation is accompanied by unfolding, and it has to be reduced and reacquire the native fold to exert its neurotoxicity. The Thioredoxin reductase-Thioredoxin system is responsible for the reduction, but it is unknown whether the refolding of L is spontaneous or aided by host chaperones. Here we report that geldanamycin, a specific inhibitor of heat shock protein 90, hampers the refolding of L after membrane translocation and completely prevents the cleavage of SNAREs. We also found that geldanamycin strongly synergises with PX-12, an inhibitor of thioredoxin, suggesting that the processes of L chain refolding and interchain disulfide reduction are strictly coupled. Indeed we found that the heat shock protein 90 and the Thioredoxin reductase-Thioredoxin system physically interact on synaptic vesicle where they orchestrate a chaperone-redox machinery which is exploited by clostridial neurotoxins to deliver their catalytic part into the cytosol.

Redox regulation of Clostridial neurotoxins into nerve terminal

Redox regulation of Clostridial neurotoxins into nerve terminal

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of invivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the invitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity‐dependent bulk endocytosis

Activity‐dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity‐dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine‐dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium‐dependent events such as activity‐dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity‐dependent dynamin I dephosphorylation was also arrested in EGTA‐treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity‐dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity‐dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

Functional evaluation of biological neurotoxins in networked cultures of stem cell-derived central nervous system neurons.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

140. Broad-spectrum detection of all clostridial neurotoxins in stem cell‒derived neurons and identification of compounds that rescue synaptic activity in synaptic function‒based assays

140. Broad-spectrum detection of all clostridial neurotoxins in stem cell‒derived neurons and identification of compounds that rescue synaptic activity in synaptic function‒based assays

Botulinum neurotoxin homologs in non-Clostridium species

Clostridial neurotoxins (CNTs) are the deadliest toxins known and the causative agents of botulism and tetanus. Despite their structural and functional complexity, no CNT homologs are currently known outside Clostridium. Here, we report the first homologs of Clostridium CNTs within the genome of the rice fermentation organism Weissella oryzae SG25. One gene in W. oryzae S25 encodes a protein with a four-domain architecture and HExxH protease motif common to botulinum neurotoxins (BoNTs). An adjacent gene with partial similarity to CNTs is also present, and both genes seem to have been laterally transferred into the W. oryzae genome from an unknown source. Identification of mobile, CNT-related genes outside of Clostridium has implications for our understanding of the evolution of this important toxin family.

Clostridial Neurotoxins: Mode of Substrate Recognition and Novel Therapy Development

The clostridial neurotoxins (CNTs) are among the most potent protein toxins known to humans. CNTs include seven serotypes (A~G) of botulinum toxins (BoNTs), which cause botulism, a flaccid paralysis, and tetanus toxin (TeNT), which causes spastic paralysis. BoNTs are classified as category A agent and may be used as potential bioterrorism weapons. On the other hand, the ability of an extremely low dosage of BoNTs (less than 1 ng) to cause reversible partial paralysis upon injection into muscle has turned BoNTs, in particular serotypes A and B, from deadly agents to novel therapeutic agents for treatment of a wide range of clinical conditions associated with involuntary muscle spasm and contractions. In addition to clinical use, they may also be used in cosmetics. Further indications for BoNTs will continue to be developed, although current BoNT therapies have encountered some limitations due to the pharmacological properties of BoNTs, such as their ability to elicit immunoresistance in patients upon periodical injections. This review summarizes the present knowledge of the mechanisms of action of CNTs, with particular focus on the mode of substrate recognition by CNT catalytic domains and knowledge regarding substrate recognition can be utilized to develop novel BoNT products to extend its usefulness in therapeutic interventions and overcome the immunoresistance problems.

Pentavalent replicon vaccines against botulinum neurotoxins and tetanus toxin using DNA-based Semliki Forest virus replicon vectors

The clostridial neurotoxin (CNT) family includes botulinum neurotoxin (BoNT), serotypes A, B, E, and F of which can cause human botulism, and tetanus neurotoxin (TeNT), which is the causative agent of tetanus. This suggests that the greatest need is for a multivalent or multiagent vaccine that provides protection against all 5 agents. In this study, we investigated the feasibility of generating several pentavalent replicon vaccines that protected mice against BoNTs and TeNT. First, we evaluated the potency of individual replicon DNA or particle vaccine against TeNT, which induced strong antibody and protective responses in BALB/c mice following 2 or 3 immunizations. Then, the individual replicon TeNT vaccines were combined with tetravalent BoNTs vaccines to prepare 4 types of pentavalent replicon vaccines. These replicon DNA or particle pentavalent vaccines could simultaneously and effectively induce antibody responses and protect effects against the 5 agents. Finally, a solid-phase assay showed that the sera of pentavalent replicon formulations-immunized mice inhibited the binding of THc to the ganglioside GT1b as the sera of individual replicon DNA or particle-immunized mice. These results indicated these pentavalent replicon vaccines could protect against the 4 BoNT serotypes and effectively neutralize and protect the TeNT. Therefore, our studies demonstrate the utility of combining replicon DNA or particle vaccines into multi-agent formulations as potent pentavalent vaccines for eliciting protective responses against BoNTs and TeNT.

611. Gene-Based Neuromodulation by Adenoviral Transduction of Light Chain Clostridial Neurotoxins

611. Gene-Based Neuromodulation by Adenoviral Transduction of Light Chain Clostridial Neurotoxins