RESUMO
The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.
Assuntos
Toxinas Botulínicas/isolamento & purificação , Botulismo/diagnóstico , DNA Bacteriano/análise , Ensaios Enzimáticos/métodos , Neurotoxinas/química , Animais , Técnicas Biossensoriais , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Botulismo/microbiologia , Clostridium botulinum/química , Clostridium botulinum/enzimologia , Clostridium botulinum/genética , Clostridium botulinum/patogenicidade , DNA Bacteriano/química , Endopeptidases/química , Ativação Enzimática , Imunoensaio/métodos , Espectrometria de Massas , Camundongos , Neurotoxinas/genética , Fatores de TempoRESUMO
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.