Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% α-helix, 50%β-sheets, 28% random coils, and 3%β-turns. This compared to 22% α-helix, 44%β-sheets, 34% random coils, and noβ-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%α-helix, 45%β-sheets, 27% random coils, and 3%β-turns, suggesting a significant alteration at least in theα-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^?1 atpH 5.7 compared to 4.0 min^?1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.     

Loop 3 of Short Neurotoxin II is an Additional Interaction Site with Membrane-bound Nicotinic Acetylcholine Receptor as Detected by Solid-state NMR Spectroscopy

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of α-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of α-bungarotoxin bound to an extracellular domain of an α-subunit of the receptor reveals different contact areas for long and short α-neurotoxins.     

High level expression,purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Botulinum neurotoxin type E heavy chain consists of two domains: N-terminal half as a translocation domain and C-terminal half (Hcc) as a binding domain. In this research a synthetic gene fragment encoding the binding domain of botulinum neurotoxin type E (BoNT/E-Hcc) was highly expressed in Escherichia coli by pGEX4T-1 vector. After purification, the recombinant BoNT/E-Hcc was evaluated by SDS-PAGE and western blot (immunoblot) analysis. Average yields obtained in this research were 3.7 mg recombinant BoNT/E-Hcc per liter of bacterial culture. The recombinant protein was injected in mice for study of its protection ability against botulinum neurotoxin type E challenges. The challenge studies showed that, vaccinated mice were fully protected against 10⁴ × minimum lethal dose of botulinum neurotoxin type E.     

Complex organizational structure of the genome revealed by genome-wide analysis of single and alternative promoters in Drosophila melanogaster

Background

Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray.

Results

Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI.

Conclusion

Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.     

Purification and Characterization of Neurotoxin Complex from a Dual Toxin Gene Containing Clostridium Botulinum Strain PS-5

Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography–Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.     

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

A Protease-Resistant Novel Hemagglutinin Purified from Type A Clostridium botulinum

Botulinum neurotoxin is a food poisoning agent produced by Clostridium botulinum. The neurotoxin is a 150-kDa protein that causes flaccid muscle paralysis by blocking neurotransmitter release at neuromuscular junctions. The neurotoxin is produced along with a group of neurotoxin associated proteins (NAPs), which protect it from the low pH and proteases of the gastrointestinal (GI) tract. We have isolated, for the first time, one of the major components of NAPs in a pure form. The isolated protein is a 33-kDa single polypeptide (Hn-33) that exhibits hemagglutination activity. Specific polyclonal antibodies against the Hn-33 are able to block the hemagglutination activity of the neurotoxin complex, which indicates that perhaps Hn-33 is the only strong hemagglutinating protein in the complex. The Hn-33 was found be resistant to trypsin and other protease digestion, a feature that could play a role in the protection of the neurotoxin in the GI tract during its toxicoinfection.     

Subunit Stoichiometry of the Clostridium botulinum Type A Neurotoxin Complex Determined Using Denaturing Capillary Electrophoresis

A denaturing capillary electrophoresis method was developed to evaluate the subunit stoichiometry of the Clostridium botulinum type A neurotoxin complex. The results indicate that the neurotoxin complex contains single copies of the 150 kDa neurotoxin and the non-toxic non-hemagglutinating subunits as well as 5–6 HA17, 4–5 HA23, 3–4 HA48, and 8–9 HA34 subunits. The calculated molecular mass for a complex with this stoichiometry is between 880 and 1,000 kDa. The molecular mass of the intact complex was determined using size-exclusion HPLC (SE-HPLC) and SE-HPLC in conjunction with multi-angle laser light scattering detection. Based on a comparison to a mixture of standard proteins, SE-HPLC analysis yielded a molecular mass of 880 kDa while light scattering analysis indicated a weight average molecular mass of 925 ± 45 kDa. The close agreement between the molecular mass values determined by the three approaches supports the subunit stoichiometry proposed for the C. botulinum type A neurotoxin complex.     

Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 10³ spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Clostridium botulinum Strain Af84 Contains Three Neurotoxin Gene Clusters: Bont/A2, bont/F4 and bont/F5

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.     

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.     

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.     

Somaclonal Variation in a Food Legume—Lathyrus sativus

Somaclones of Lathyrus sativus cv P-24 were obtained from leaf, internode and root derived callus cultures. These showed significantly decreased neurotoxin (ODAP) content up to 0.03% compared to that of parent cultivar P-24 (0.3%). The somaclones also showed higher seed yield than parent P24. In addition. somaclone Bio 1–22 showed significantly decreased time for flowering. Other heritable morphological variant features were leaf length, leaf breadth, internode length, flower colour, seed colour, 100 seed weight, neurotoxin content of leaves and red markings on the pods.     

Geographical Segregation of the Neurotoxin-Producing Cyanobacterium Anabaena circinalis

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.