title: 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.
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Skadi
authors: S
authors: Kull
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: K Melanie
authors: KM
authors: Schulz
authors: Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors: Jasmin
authors: J
authors: Weisemann
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Sebastian
authors: S
authors: Kirchner
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Tanja
authors: T
authors: Schreiber
authors: Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors: Alexander
authors: A
authors: Bollenbach
authors: Highly Pathogenic Viruses (ZBS1), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: P Wojtek
authors: PW
authors: Dabrowski
authors: Highly Pathogenic Viruses (ZBS1), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Andreas
authors: A
authors: Nitsche
authors: Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia, United States of America.
authors: Suzanne R
authors: SR
authors: Kalb
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Martin B
authors: MB
authors: Dorner
authors: Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, Atlanta, Georgia, United States of America.
authors: John R
authors: JR
authors: Barr
authors: Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors: Andreas
authors: A
authors: Rummel
authors: Biological Toxins (ZBS3), Centre for Biological Threats and Special Pathogens, Robert Koch-Institut, Berlin, Germany.
authors: Brigitte G
authors: BG
authors: Dorner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D001906:Botulism / Q000453:epidemiology* / Q000382:microbiology* / Q000473:pathology; D047013:Clostridium botulinum type A / Q000235:genetics*; D004196:Disease Outbreaks; D057141:Food, Preserved / Q000382:microbiology; D005858:Germany / Q000453:epidemiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D012336:RNA, Ribosomal, 16S; D019274:Botulinum Toxins, Type A