A Cell Line for Detection of Botulinum Neurotoxin Type B

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines

Onset dynamics of type A botulinum neurotoxin-induced paralysis

Experimental studies have demonstrated that botulinum neurotoxin serotype A (BoNT/A) causes flaccid paralysis by a multi-step mechanism. Following its binding to specific receptors at peripheral cholinergic nerve endings, BoNT/A is internalized by receptor-mediated endocytosis. Subsequently its zinc-dependent catalytic domain translocates into the neuroplasm where it cleaves a vesicle-docking protein, SNAP-25, to block neurally evoked cholinergic neurotransmission. We tested the hypothesis that mathematical models having a minimal number of reactions and reactants can simulate published data concerning the onset of paralysis of skeletal muscles induced by BoNT/A at the isolated rat neuromuscular junction (NMJ) and in other systems. Experimental data from several laboratories were simulated with two different models that were represented by sets of coupled, first-order differential equations. In this study, the 3-step sequential model developed by Simpson (J Pharmacol Exp Ther 212:16–21,1980) was used to estimate upper limits of the times during which anti-toxins and other impermeable inhibitors of BoNT/A can exert an effect. The experimentally determined binding reaction rate was verified to be consistent with published estimates for the rate constants for BoNT/A binding to and dissociating from its receptors. Because this 3-step model was not designed to reproduce temporal changes in paralysis with different toxin concentrations, a new BoNT/A species and rate (kS) were added at the beginning of the reaction sequence to create a 4-step scheme. This unbound initial species is transformed at a rate determined by kS to a free species that is capable of binding. By systematically adjusting the values of kS, the 4-step model simulated the rapid decline in NMJ function (kS ≥0.01), the less rapid onset of paralysis in mice following i.m. injections (kS = 0.001), and the slow onset of the therapeutic effects of BoNT/A (kS < 0.001) in man. This minimal modeling approach was not only verified by simulating experimental results, it helped to quantitatively define the time available for an inhibitor to have some effect (tinhib) and the relation between this time and the rate of paralysis onset. The 4-step model predicted that as the rate of paralysis becomes slower, the estimated upper limits of (tinhib) for impermeable inhibitors become longer. More generally, this modeling approach may be useful in studying the kinetics of other toxins or viruses that invade host cells by similar mechanisms, e.g., receptor-mediated endocytosis

Light Chain Separated from the Rest of the Type A Botulinum Neurotoxin Molecule Is the Most Catalytically Active Form

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as “belt,” that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that Km for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (kcat) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo

Clostridial Neurotoxins: Mechanism of SNARE Cleavage and Outlook on Potential Substrate Specificity Reengineering

The clostridial neurotoxin family consists of tetanus neurotoxin and seven distinct botulinum neurotoxins which cause the diseases tetanus and botulism. The extreme potency of these toxins primarily relies not only on their ability to specifically enter motoneurons but also on the activity their catalytic domains display inside presynaptic motoneuronal terminals. Subsequent to neurotoxin binding and endocytosis the catalytic domains become translocated across endosomal membranes and proteolyze unique peptide bonds of one of three soluble N-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs), vesicle associated membrane protein/synaptobrevin, synaptosome associated protein of 25 kDa, or syntaxin. As these substrate proteins are core components of the vesicular membrane fusion apparatus, cleavage of any of the substrate molecules results in the blockade of neurotransmitter release. This review summarizes the present knowledge about the molecular basis of the specific substrate recognition and cleavage mechanism and assesses the feasibility of reengineering catalytic domains to hydrolyze non-substrate members of the three SNARE families in order to expand the therapeutic application of botulinum neurotoxins

Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach

Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme

Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication

Tetanus toxin Hc fragment induces the formation of ceramide platforms and protects neuronal cells against oxidative stress

Tetanus toxin (TeTx) is the protein, synthesized by the anaerobic bacteria Clostridium tetani, which causes tetanus disease. TeTx gains entry into target cells by means of its interaction with lipid rafts, which are membrane domains enriched in sphingomyelin and cholesterol. However, the exact mechanism of host membrane binding remains to be fully established. In the present study we used the recombinant carboxyl terminal fragment from TeTx (Hc-TeTx), the domain responsible for target neuron binding, showing that Hc-TeTx induces a moderate but rapid and sustained increase in the ceramide/sphingomyelin ratio in primary cultures of cerebellar granule neurons and in NGF-differentiated PC12 cells, as well as induces the formation of ceramide platforms in the plasma membrane. The mentioned increase is due to the promotion of neutral sphingomyelinase activity and not to the de novo synthesis, since GW4869, a specific neutral sphingomyelinase inhibitor, prevents neutral sphingomyelinase activity increase and formation of ceramide platforms. Moreover, neutral sphingomyelinase inhibition with GW4869 prevents Hc-TeTx-triggered signaling (Akt phosphorylation), as well as the protective effect of Hc-TeTx on PC12 cells subjected to oxidative stress, while siRNA directed against nSM2 prevents protection by Hc-TeTx of NSC-34 cells against oxidative insult. Finally, neutral sphingomyelinase activity seems not to be related with the internalization of Hc-TeTx into PC12 cells. Thus, the presented data shed light on the mechanisms triggered by TeTx after membrane binding, which could be related with the events leading to the neuroprotective action exerted by the Hc-TeTx fragment

Bacterial Toxins and the Nervous System: Neurotoxins and Multipotential Toxins Interacting with Neuronal Cells

Toxins are potent molecules used by various bacteria to interact with a host organism. Some of them specifically act on neuronal cells (clostridial neurotoxins) leading to characteristics neurological affections. But many other toxins are multifunctional and recognize a wider range of cell types including neuronal cells. Various enterotoxins interact with the enteric nervous system, for example by stimulating afferent neurons or inducing neurotransmitter release from enterochromaffin cells which result either in vomiting, in amplification of the diarrhea, or in intestinal inflammation process. Other toxins can pass the blood brain barrier and directly act on specific neurons

Tight correlation between inhibition of DNA repair in vitro and transcription factor IIIA binding in a 5S ribosomal RNA gene

UV‐induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)–5S rDNA complex and Xenopus oocyte nuclear extracts. TFIIIA alters CPD formation primarily in the transcribed strand of the 50 bp internal control region (ICR) of 5S rDNA. During NER in vitro, CPD removal is reduced at most sites in both strands of the ICR when TFIIIA is bound. Efficient repair occurs just outside the TFIIIA‐binding site (within 10 bp), and in the absence of 5S rRNA transcription. Interestingly, three CPD sites within the ICR [+56, +75 (transcribed strand) and +73 (non‐transcribed strand)] are repaired rapidly when TFIIIA is bound, while CPDs within ∼5 bases of these sites are repaired much more slowly. CPDs at these three sites may partially displace TFIIIA, thereby enabling rapid repair. However, TFIIIA is not completely displaced during NER, at least at sites outside the ICR, even though the NER complex could be sterically hindered by TFIIIA. Such inefficient repair of transcription factor binding sites could increase mutation frequency in regulatory regions of genes

Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies.

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans