title: Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster.
abstract: BACKGROUND
  Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.
  
  
  RESULTS
  The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.
  
  
  CONCLUSIONS
  Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.
authors.affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada. acdoxey@uwaterloo.ca
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Doxey
authors.forename: Michael D J
authors.initials: MD
authors.lastname: Lynch
authors.forename: Kirsten M
authors.initials: KM
authors.lastname: Müller
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Meiering
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: McConkey
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics*; D003014:Clostridium botulinum / Q000235:genetics*; D017364:Collagenases / Q000737:chemistry / Q000235:genetics; D022801:Complementarity Determining Regions / Q000235:genetics; D019143:Evolution, Molecular; D005408:Flagellin / Q000737:chemistry / Q000235:genetics; D020440:Gene Duplication; D023061:Gene Order; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D022801:Complementarity Determining Regions; D009498:Neurotoxins; D005408:Flagellin; D017364:Collagenases; D001905:Botulinum Toxins