Enzymatic breakdown of tetanus toxin

Treatment of tetanus toxin with papain at 55°C resulted in breakdown of the molecule to yield an atoxic fraction with a molecular weight of approximately 40 000. The highly purified material exhibited partial immunological identity with the parent toxin, showed no toxicity and elicited the formation of neutralizing antibodies against tetanus.     

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli.

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.     

Retrograde axonal transport of an exogenous enzyme covalently linked to B-IIb fragment of tetanus toxin.

Attempt to replace enzymes in a number of fatal lysosomal storage disease involving the central nervous system have as yet been unsuccessful owing to the impermeability of the blood/brain barrier to macromolecules. In order to treat storage disease due to enzyme deficiencies, we investigated the feasibility of transporting an enzyme into the central nervous system without crossing the blood/brain barrier. Using the B-IIb fragment of tetanus toxin (because it is involved in recognition by the nerve-cell endings), retrograde axonal transport toward the spinal cord and trans-synaptic movement, and glucose oxidase as a marker, we demonstrated that a non-toxic enzyme-vector conjugate was taken up by axon terminals. After injection into the gastrocnemius muscle, the B-IIb-glucose oxidase conjugate was detected, both histologically and electrochemically, distally to a ligature on the sciatic nerve. Thus the B-IIb fragment could serve as a vector for glucose oxidase transport into the central nervous system. It was also verified that the transported enzyme retained its activity. Transport of this 150 kDa molecule by fragment B-IIb of tetanus toxin suggests that other enzymes of a lesser molecular mass may also be transported.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.     

Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin.

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.     

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.     

破伤风毒素C片段(TTc)在大肠杆菌中的表达、优化与鉴定

HTSS以一株破伤风生产菌株基因组DNA为模板,通过上游引物中几个碱基的修改,PCR扩增出破伤风毒素C片段(TTc)基因,构建了原核表达质粒pET-42(b)/TTc,在大肠杆菌BL21(DE3)中表达。重组蛋白分子量约50kD,表达量为22%,超声波破碎显示为可溶性重组蛋白。通过对培养基、诱导时间、诱导温度的优化,重组蛋白的表达量和可溶性均有提高。Western blotting检测表达产物可与破伤风C片段单克隆抗体产生特异的免疫反应。该工作为亚单位疫苗或载体蛋白的开发奠定了基础。     

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

Abstract The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 × LD₅₀) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10–20 fold) more immunogenic than the alternative forms of the protein.     

Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge

To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) In L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the Immune system in an immunogenic form.     

Analysis of mutants of tetanus toxin Hc fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Identification and characterization of an RTX toxin in the emerging pathogen Kingella kingae

Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).     

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.     

Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.

Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.