Isolation and Functional Characterization of the Novel Clostridium botulinum Neurotoxin A8 Subtype

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins

Differential binding of antibodies to BoNT/A1 and BoNT/A8.

<p>Native or recombinantly expressed single chain BoNT/A1 and single chain BoNT/A8 were coated onto microtiter plates and tested in an indirect ELISA for capturing a panel of monoclonal and polyclonal antibodies generated in-house. All antibodies shown are monoclonal antibodies except for AR47 and AC29 which are polyclonal antibodies derived from rabbit and chicken, respectively. Shown is the mean (± SD) of three independent experiments performed in duplicates.</p

Binding characteristics of BoNT/A8 cell-binding domain H_C.

<p>(A) Binding affinity of H_CA8 to its synaptic protein receptor rat SV2C in a GST pull-down assay is similar to H_CA1. 12.5% SDS-PAGE and Coomassie staining of a representative pull-down experiment. (B) Densitometric quantification of bound H_C-fragments from (A) (n = 4 ± SD). (C) Competition assay: Binding of ³⁵S-H_CA1 to synaptosomes at 4°C in the presence of increasing amounts of recombinant H_CA1 or H_CA8, respectively. Bound ³⁵S-H_CA1 was detected by SDS-PAGE and autoradiography (n = 3 ± SD; H_CA1: EC₅₀ = 24 nM (95% CI 20–29 nM), H_CA8: EC₅₀ = 56 nM, 95% CI 43–72 nM)).</p

Comparison of the catalytic activity of BoNT/A1 and BoNT/A8.

<p>(A) Quantitative mass spectrometric endopeptidase assay of native full-length BoNT/A1 and BoNT/A8 immunoenriched from cell culture supernatants tested for cleavage of a SNAP-25 peptide substrate. (B) Quantitative mass spectrometric endopeptidase assay of recombinant light chains of BoNT/A1 and BoNT/A8 tested for cleavage of human H6SNAP-25 protein. Both diagrams show the percentage of BoNT/A8 substrate cleavage compared to BoNT/A1 (n = 3 ± SD). (C) Time-dependent cleavage of rat SNAP-25H6 (3 μM) by LC/A1 and LC/A8 (0.5 nM). The amount of cleavage product was quantified densitometrically from SDS-PAGE stained by Coomassie (n = 3 ± SD).</p

Biological activity of BoNT/A1 and A8.

<p>The biological activity of recombinant full-length active BoNT/A1 and BoNT/A8 was determined employing the mouse phrenic nerve hemidiaphragm (MPN) assay. To a three-point dose—response curve (n ≥ 3 ± SD) of each subtype a power function was fitted. To yield an identical paralytic halftime, six-fold higher concentrations of BoNT/A8 are required.</p

Surface representation of BoNT/A8 and arginine insertion.

<p>(A) Surface representation of BoNT/A (PDB code 3BTA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116381#pone.0116381.ref096" target="_blank">96</a>]). Differences of BoNT/A8 to BoNT/A1 are marked in yellow, unique amino acid differences in BoNT/A8 (based on the representatives of BoNT/A2 to A7 used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116381#pone.0116381.g001" target="_blank">Fig. 1</a>) are marked in red, and the arginine insertion in position 888 is marked in green. (B) Representative MS/MS spectra of the precursor ion m/z 923.01 ([M+2H]²⁺], inset) of BoNT/A8 with the sequence of ⁸⁷²NITNTSILSIVVDKDGR⁸⁸⁸, showing the additional arginine at position 888 leading to an additional trypsin cleavage site.</p

<i>Bont/a8</i> gene cluster organization.

<p>Schematic representation of the neurotoxin gene clusters from the prototype BoNT/A1 strain ATCC 3502, BoNT/A1(B) strain NCTC 2916, BoNT/A8 strain Chemnitz, and BoNT/F1 strain Langeland. The <i>bont/a1</i> gene from ATCC 3502 is located within an <i>ha</i>⁺<i>orfX</i>^-cluster containing the <i>non-toxic non-haemagglutinin</i> (<i>ntnha</i>) gene, the three <i>haemagglutinin</i> genes (<i>ha70</i>, <i>ha17</i> and <i>ha33</i>) and the regulatory <i>botR</i> gene. The <i>bont/a1</i> neurotoxin gene cluster of ATCC 3502 is flanked by the <i>flagellin</i> (<i>fla</i>) gene and an insertion sequence element (IS). In contrast to the prototype <i>bont/a1</i> (ATCC 3502), the <i>bont/a1</i> of NCTC 2916 and the <i>bont/a8</i> gene of Chemnitz are located in an <i>ha</i>^-<i>orfX</i>⁺ cluster which contains the adjacent <i>ntnha</i> gene and—instead of the <i>ha</i> genes—the three <i>orfX1–3</i> genes; apart from the <i>botR</i> gene the cluster also contains the <i>p47</i> gene of unknown function. The neurotoxin gene clusters of NCTC 2916 and Chemnitz are located within the ars operon on the chromosome and are flanked by the <i>arsC</i> and the <i>lycA</i> genes. Except for the toxin gene itself, its gene cluster organization and sequence is most similar to the <i>bont/f1</i> gene cluster of strain Langeland.</p

Comparison of BoNT/A subtypes.

<p>BoNT/A1 amino acid sequence of strain ATCC 3502 as prototype was compared to representatives of subtypes BoNT/A2 to A7 and the novel BoNT/A8. Sequence differences are indicated by vertical lines. The cartoon indicates the domain of the light chain (LC), the <i>N</i>-terminal part (H_N) and the <i>C</i>-terminal part (H_C) of the heavy chain (HC). The latter can be further subdivided into an <i>N</i>-terminal (H_CN) and <i>C</i>-terminal (H_CC) subdomain. The alignment illustrates that the majority of differences between BoNT/A8 and A1 are located in the heavy chain. Furthermore, the arginine insertion at position 888 is unique to BoNT/A8.</p

Identity of BoNT/A8 to other BoNT/A subtypes on nucleic acid and amino acid levels.^*

<p>* Shown is the percentage of nucleic acid (nt) and amino acid (aa) identity among the subtypes BoNT/A1 to A8 subdivided into light chain (LC), <i>N</i>-terminal domain (H_N) and <i>C</i>-terminal domain (H_C) of the heavy chain (HC) and of the holotoxin. Additionally, the <i>N</i>-terminal (H_CN) and <i>C</i>-terminal (H_CC) subdomains of H_C are displayed. Highest percentage of identity (±0.5) is depicted in <b>bold</b> and lowest percentage of identity (±0.5) is depicted in <i>italic</i>.</p><p>Identity of BoNT/A8 to other BoNT/A subtypes on nucleic acid and amino acid levels.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116381#t001fn001" target="_blank">*</a>}</p

Sequence identity of individual genes within the neurotoxin gene cluster of strain Chemnitz encoding BoNT/A8 compared to other BoNT/A, E and F sero- and subtypes located in an <i>ha</i>^-<i>orfx</i>⁺ gene cluster on nucleotide levels.^*

<p>* The neurotoxin gene clusters encoding for BoNT, NTNHA, p47, BotR and the OrfX1, OrfX2 and OrfX3 proteins were compared between strain Chemnitz (BoNT/A8) and the other BoNT/A subtypes (A1 to A7) and percent identities are given either on nucleic acid level (A) or on amino acid level (B). For comparison the identities to BoNT/E and BoNT/F also located within an <i>ha</i>^-<i>orfX</i>⁺ neurotoxin gene cluster are displayed, too. Highest identities (±0.5) are depicted in <b>bold</b> and lowest identities (±0.5) are depicted in <i>italic</i>. Empty cells indicate that no sequence data exist while n/a denotes the absence of certain genes from the neurotoxin gene cluster.</p><p>Sequence identity of individual genes within the neurotoxin gene cluster of strain Chemnitz encoding BoNT/A8 compared to other BoNT/A, E and F sero- and subtypes located in an <i>ha</i>^-<i>orfx</i>⁺ gene cluster on nucleotide levels.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116381#t002fn001" target="_blank">*</a>}</p