Characterisation of botulinum toxins type C, D, E, and F by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

Abstract

In a follow-up of the earlier characterisation of botulinum toxins type A and B (BTxA and BTxB) by mass spectrometry (MS), types C, D, E, and F (BTxC, BTxD, BTxE, BTxF) were now investigated. Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxC, BTxD, BTxE, and BTxF are comprised of a protein complex of the respective neurotoxins with non-toxic non-haemagglutinin (NTNH) and, sometimes, specific haemagglutinins (HA). These protein complexes were observed in mass spectrometric identification. The BTxC complex, from Clostridium botulinum strain 003-9, consisted of a 'type C1 and D mosaic' toxin similar to that of type C strain 6813, a non-toxic non-hemagglutinating and a 33 kDa hemagglutinating (HA-33) component similar to those of strain C-Stockholm, and an exoenzyme C3 of which the sequence was in full agreement with the known genetic sequence of strain 003-9. The BTxD complex, from C. botulinum strain CB-16, consisted of a neurotoxin with the observed sequence identical with that of type D strain BVD/-3 and of an NTNH with the observed sequence identical with that of type C strain C-Yoichi. Remarkably, the observed protein sequence of CB-16 NTNH differed by one amino acid from the known gene sequence: L859 instead of F859. The BTxE complex, from a C. botulinum isolated from herring sprats, consisted of the neurotoxin with an observed sequence identical with that from strain NCTC 11219 and an NTNH similar to that from type E strain Mashike (1 amino acid difference with observed sequence). BTxF, from C. botulinum strain Langeland (NCTC 10281), consisted of the neurotoxin and an NTNH; observed sequences from both proteins were in agreement with the gene sequence known from strain Langeland. As with BTxA and BTxB, matrix-assisted laser desorption/ionisation (MALDI) MS provided provisional identification from trypsin digest peptide maps and liquid chromatography-electrospray (tandem) mass spectrometry (LC-ES MS) afforded unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin digestion.

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

“…In our study, we analyzed complete progenitor toxins from serotypes A through G. Five of the strains we used in this study differed from those analyzed by van Baar et al (29,30), who used strains 62A, Okra, 003-9, CB-16, no designation (equivalent to NCTC 11219 by analysis), and Langeland from serotypes A through F, respectively (30). Okra and Langeland strains from serotypes B and F, respectively, overlapped those used in this study.…”

“…Their MALDI-MS and LC-ESI-MS analyses of botulinum serotypes A through F identified neurotoxin and each protein present in their toxin samples. They also showed that ESI-MS with CID (ESI-MS/MS) or MALDI-MS with postsource decay identified protein unambiguously (29,30). Several neurotoxin samples they purchased included some known progenitor proteins, but they did not always include each progenitor toxin protein in each sample.…”

“…van Baar et al proposed that protein toxins can be unambiguously identified with mass spectrometry (29), and they applied this premise to the analyses of tetanus (28) and botulinum toxins (29,30). Their MALDI-MS and LC-ESI-MS analyses of botulinum serotypes A through F identified neurotoxin and each protein present in their toxin samples.…”

“…Two major concerns regarding progenitor toxin strain assignment involve the availability of nucleotide/amino acid sequence information for individual C. botulinum strains and the level of amino acid sequence identity among NAPs from different strains. In recent reports, van Baar et al (29,30) addressed some of these issues when they characterized neurotoxin samples from serotypes A (strain 62A), B (strain Okra), C (003-9), D (CB-16), E (no designation; equivalent to NCTC 11219 by analysis), and F (Langeland) that also contained some progenitor toxin proteins. They analyzed these samples with matrix-assisted laser desorption ionization (MALDI)-MS and capillary LC-ESI-MS methods originally developed for tetanus toxin analysis (28).…”

“…Progenitor toxin size distributions vary by serotype, with type A strains producing 19S, 16S, and 12S progenitor toxins, type B, C, D, and E strains producing 16S and 12S progenitor toxins, and type F and G strains producing only 12S and 16S progenitor toxins, respectively (9,17,22,23,24,31,34). Neurotoxin and NTNH comprise the 12S (M) progenitor toxin (30), the 16S (L) progenitor toxin is composed of neurotoxin, NTNH, and additional NAPs (10,15,19), and the 19S (LL) progenitor toxin is a dimer of the 16S (L) progenitor toxin. Genes encoding progenitor toxins are clustered, have a defined spatial order, and are expressed in a coordinated manner, and progenitor toxin assembly apparently proceeds in a concerted fashion (6,7,14,16,18).…”