title: Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.
abstract: Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.
authors.affiliation: Centers for Disease Control and Prevention , National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, NE, Atlanta, Georgia 30341, United States.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Kalb
authors.forename: Jakub
authors.initials: J
authors.lastname: Baudys
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Smith
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000145:classification / Q000494:pharmacology*; D004798:Enzymes / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry / Q000145:classification / Q000494:pharmacology*; D020033:Protein Isoforms / Q000737:chemistry / Q000145:classification / Q000494:pharmacology; D040901:Proteomics; D017386:Sequence Homology, Amino Acid
chemical_list: D004798:Enzymes; D009498:Neurotoxins; D020033:Protein Isoforms; D001905:Botulinum Toxins