During a food-borne botulism outbreak affecting a 63-year-old male near the city of Chemnitz in December 2007, suspected food samples (salad of home-canned green beans and two different types of sausages) were investigated. The green beans were found positive by multiplex qPCR for bont/a prior and after anaerobic enrichment culture. Subsequently, the C. botulinum strain "Chemnitz" was isolated.
Sequencing of the 16S rRNA and bont/a genes of strain Chemnitz confirmed the identity of a BoNT/A producing C. botulinum group I strain and revealed that the bont/a gene sequence differed from those published by more than 2.6%, indicating that it might be a novel subtype (Table 1). The novel bont/a gene showed highest identity to bont/a1, a5 and a6 (96.2-96.6%). On protein level, the translated sequence differed by at least 6.6% from BoNT/A2 and A5 (Table 1). Most distant from all currently known BoNT/A subtypes was BoNT/A3 with 6.9% and 12.3% difference on nucleotide and amino acid level, respectively. The novel BoNT/A subtype of strain Chemnitz was designated BoNT/A8, the next consecutive subtype number.
 Fig. 1 shows a schematic representation of BoNT/A1 to BoNT/A8, with vertical lines representing differences in amino acids compared to BoNT/A1. The alignment illustrates that most differences between BoNT/A1 and BoNT/A8 are located on the heavy chain of the molecule. A unique feature of BoNT/A8 was a triplet insertion (AGG) at position 2662, leading to an additional arginine at amino acid position 888. This makes BoNT/A8 the second 1297 aa long BoNT/A subtype next to the BoNT/A2 of strain CDC2171 (Fig. 1 and S1 Fig.). Overall, BoNT/A8 showed 87 amino acid mutations compared to BoNT/A1, with 12 amino acid substitutions at positions being unique for BoNT/A8 (Fig. 2A and S1 Fig.).
We compared the identities of the four domains and subdomains of BoNT/A8 to other BoNT/A subtypes and also to BoNT/E and BoNT/F as closest relatives (Table 1). The identities for the domains and subdomains vary greatly. E.g. whereas the holotoxin and LC of BoNT/A8 had the lowest identity towards BoNT/A3 (Loch Maree), the identity of the HCN subdomain between BoNT/A8 and BoNT/A3 was one of the highest. Conversely, BoNT/A1 showed the highest identity to BoNT/A8 for the LC and HN domains whereas the HCN subdomain differed the most.
BoNT/A is the only serotype encoded either in an ha + orfX -or an ha - orfX + neurotoxin gene cluster. To elucidate the organization of the neurotoxin gene cluster of strain Chemnitz, we applied whole genome shotgun sequencing (454 sequencing on a GS FLX+ System, Roche). Additionally, the gene cluster was partly verified by Sanger sequencing and is available under GenBank accession number KM233166. Noteworthy, Sanger sequencing of the bont/a gene with the primers published by Hill et al. resulted in a different last amino acid as compared to the 454 shot gun sequence (L Q). This is based on the fact that the Sanger sequence was forced by the primer 6R sequence to CTG (encoding L) instead of the CAG (encoding Q) which was unambiguously obtained by whole genome sequencing. The bont/a8 cluster belonged to the ha - orfX + type (Fig. 3) which is known to encode BoNT/A1, A1(B), A2, A3, A4, and A6 subtypes as well as BoNT/E and F serotypes. The bont/a8 gene cluster was located within the ars operon on the chromosome like other ha - orfX + BoNT/A- and BoNT/F-producing strains of group I and is flanked by the arsC and the lycA genes. Noteworthy, the whole bont/a8 gene cluster:with the exception of the bont/a8 gene itself:was closest related to the bont/f1 gene cluster of strain Langeland also belonging to C. botulinum group I (Fig. 3, Table 2 and Table 3).
In addition to genomic characterization, the protein sequence of BoNT/A8 was analyzed by high resolution LC-MS/MS. After immunoaffinity enrichment the BoNT was digested with three endopeptidases (AspN, trypsin and chymotrypsin), leading to a sequence coverage of ~67%. MS/MS spectra of the doubly charged precursor ion m/z 923.01 revealed the amino acid sequence 872NITNTSILSIVVDKDGR888 and verified the arginine insertion at position 888 (Fig. 2B), which led to an additional trypsin cleavage site. Additionally, 57 out of 87 amino acid mutations deduced from nucleotide sequence could be confirmed by this protein sequencing approach (S2 Fig.).
Furthermore, the MS/MS data of the novel subtype were used to challenge the amino acid substitution database recently described. The amino acid substitution database identified nine of the 12 unique point mutations in BoNT/A8 compared to the sequence of BoNT/A1 and the other known subtypes (S1 and S2 Figs.). On this basis the substitution database classified BoNT/A8 as "novel" subtype.
We tested whether the observed differences on protein level resulted in an altered antibody recognition pattern: both BoNT/A8 and BoNT/A1 were recombinantly expressed as single-chain molecules, immobilized on a 96-well plate and tested for binding a panel of monoclonal and polyclonal antibodies generated in-house ( and unpublished data). As shown in Fig. 4, a number of monoclonal antibodies (mAbs) showed no difference in recognizing BoNT/A1 and A8, while others showed a reduced binding or even a complete loss of binding to BoNT/A8 (mAb HcA16/6, HcA78/6, HcA86/2, A324/10, and A401/3).
The antibodies showing a differential binding between BoNT/A1 and BoNT/A8 recognize different epitopes on the BoNT molecule, with mAb HcA16/6, HcA78/6 and HcA86/2 being directed against the HC-domain, mAb A401/3 recognizing the LC domain and mAb A324/10 recognizing a yet to be determined conformational epitope on the molecule. Binding to two polyclonal antibodies raised in rabbit (AR47) or chicken (AC29) was not affected (Fig. 4).
Little is known about the functional consequences of the sequence differences of BoNT subtypes. Most of our knowledge regarding binding of BoNT to the synaptic membrane, transcytosis and proteolytic action towards SNAP-25 is based on the archetype BoNT/A1. In our first set of experiments we investigated if differences in the amino acid sequence affect binding to the synaptic cell surface. Binding of BoNT/A follows a two-receptor model in which BoNT/A shows a low affinity interaction towards gangliosides and a high affinity binding to synaptic vesicle glycoprotein 2 (SV2) family members. BoNT/A1 displays the highest affinity to the luminal domain 4 of SV2 isoform C. First we analyzed binding of the recombinant isolated cell binding domain HC to its immobilized protein receptor rat SV2C. The corresponding 126 aa peptide 454-579 has been fused to glutathione-S-transferase (GST) to allow immobilization on GT-matrix. GST-rSV2C 454-579 could equally well precipitate HCA1 and HCA8 from the supernatant, indicating no change in affinity towards their protein receptor (Figs. 5A and 5B). We then measured the interaction with gangliosides by the interaction of the HC-domain with synaptosomes at 4 C. In contrast to an equal interaction with the protein receptor, the binding of radiolabeled 35S-HCA1 to synaptosomes was inhibited by lower concentrations of recombinant HCA1 than those of HCA8. Whereas recombinant HCA1 displayed an EC50 of 24 nM (95% CI 20-29 nM), 2.5-fold more HCA8 was required to displace 35S-HCA1 (EC50 = 56 nM, 95% CI 43-72 nM), indicating that HCA8 has a lower binding affinity to gangliosides incorporated in neuronal membranes compared to HCA1 (Fig. 5C).
After characterization of the binding domain we explored the LC for differences in its catalytic activity. In order to analyze the endopeptidase activity of BoNT/A8 in comparison to BoNT/A1, a combination of immunoaffinity enrichment plus mass spectrometric quantification of substrate cleavage was performed. The native toxins were enriched from cell-free culture supernatants using mAb 4E17.1 coupled to magnetic beads which recognizes the strictly conserved epitope YNQYTEEEK (aa 751-759; and S1 Fig.). Captured toxins were then incubated in an Endopep-MS assay with a cleavable 22-mer SNAP-25 peptide. We found that BoNT/A8 showed only about 28% activity compared to that of BoNT/A1 (Fig. 6A). It has been described that the antibody used for immunocapturing the BoNT might affect the catalytic activity of LC in a subtype-dependent manner. To exclude any deleterious effects of the antibody used, we omitted the immunocapture step and compared the activity of recombinantly expressed LC only. LC of BoNT/A1 and BoNT/A8 were incubated with the full-length human SNAP-25 protein, and catalytic activity was determined in an Endopep-MS assay. Consistent with the results obtained with the native full-length molecule we found a dramatic reduction in catalytic activity to about 10% when comparing the activity of LC/A8 to that of LC/A1 (Fig. 6B).
Moreover, the findings of the Endopep-MS assay could be confirmed in an independent experimental setting measuring rat SNAP-25 wild-type cleavage in a time-dependent endopeptidase assay, where the cleavage activity was determined by SDS-PAGE and densitometric quantification of the cleavage product. Again, results clearly corroborated the fact that LC/A8 displays a lower rate of catalysis than LC/A1 (Fig. 6C).
To verify whether the altered characteristics in binding and catalytic activity results in a modified biological activity, the potency of full-length BoNT/A1 and BoNT/A8 molecules:recombinantly expressed as single-chain molecules and activated in vitro:was determined employing the MPN assay which represents the physiological site of action of BoNT. Comparison of the three-point dose:response curves (Fig. 7) yielded an about six-fold reduced biological activity of BoNT/A8 versus BoNT/A1, caused by the reduced binding and catalytic properties of BoNT/A8.