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

Abstract

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.

Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

“…Interestingly, this loop is not conserved among BoNT/A subtypes. In BoNT/A8, this loop is longer than that in other subtypes because of a unique arginine insertion that makes BoNT/A8 the longest BoNT/A subtype [29]. The third notable difference is that a 3/10-helix (residues S955-S957) linking β8 and β9 in H C A1 is unstructured in H C HA due to the deletion of two residues in this region (Figure 1B, yellow box; Figure 1D).…”

“…Nevertheless, it is reasonable to speculate that the ganglioside-binding profile of BoNT/HA is more similar to BoNT/A8 than BoNT/A1. It is noteworthy that H C A8 has a weaker binding affinity to gangliosides on neuronal membranes compared to H C A1, which is possibly due to the negative influence of this altered loop [29]. Thus, we suspect that the reduced ganglioside-binding ability of BoNT/HA could partly contribute to its ~5-fold lower toxicity compared to BoNT/A1, as revealed by a mouse bioassay [21].…”

Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons

“…BoNT serotypes include subtypes neutralized by serotype-specific antisera (15)(16)(17). Informatics has classified eight BoNT/A subtypes (A1 to A8), which vary by ϳ10 to 15% in amino acid identity (15,16,(18)(19)(20)(21).…”

“…Informatics has classified eight BoNT/A subtypes (A1 to A8), which vary by ϳ10 to 15% in amino acid identity (15,16,(18)(19)(20)(21). While BoNT/A1 and BoNT/A2 cleave SNAP25 with similar kinetics (22)(23)(24) and possess similar potencies in the mouse model of botulism (BoNT/A1 and BoNT/A2 had specific activities of 1.25 ϫ 10 8 and 1.27 ϫ 10 8 50% lethal doses [LD 50 ] [in U/mg], respectively, in mice) (25,26), BoNT/A1 and BoNT/A2 elicit different paralytic symptoms in mice (24).…”

On-site detection of bioterrorism-relevant agents

“…Hierdurch war gereinigtes Toxin, das weiterhin toxisch ist, nicht nachweisbar, da die in ungereinigten Präpa-rationen ebenfalls vorhandenen Proteine durch den Reinigungsschritt entfernt wurden. Solch falsch-negative Ergebnisse können ebenfalls bei Toxinvarianten auftreten, falls Antikörper hoher Spezifität Varianten aufgrund von Veränderungen an der Antikörper-Bindungsstelle nicht mehr binden können [28,29].…”