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 MSE 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.

Functional Characterization of Botulinum Neurotoxin Serotype H as a Hybrid of Known Serotypes F and A (BoNT F/A)

“…The mixtures were incubated overnight at room temperature with no mixing. The biotinylated mAbs were bound to streptavidin beads as previously described 19 .…”

“…To positively identify the novel botulinum neurotoxin extracted from the culture supernatant of CDC69016, the toxin was digested with trypsin and chymotrypsin following the same protocol previously used 19 . Peptides from the toxin were identified by LC-MS/MS with database searching as described previously 19 .…”

Historical and current perspectives on Clostridium botulinum diversity

“…However, they could not distinguish whether this toxic activity was due to "a single molecular species or if it is a mixture of types A and F toxins" [48]. It was later determined that this strain produces three distinct toxins, BoNT/A2 and two type F toxin variants (BoNT/F4 and/F5) [77]. Further examples of strains that appeared to produce mixtures containing multiple toxin types included a C. botulinum BoNT/Ba-producing strain [60], a BoNT/AB-producing strain [102], and a BoNT/Bf-producing strain [59].…”

Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q⁵⁸-K⁵⁹ Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities

“…The so far examined BoNT/F subtypes F1, F2, F4, F6, and F7 have been shown to cleave the VAMP-2 peptide bond Q 58 -K 59 [15][16][17][18][19], whereas BoNT/F5 hydrolyzes VAMP-2 at L 54 -E 55 [11].…”

“…All subtypes studied so far share the scissile substrate peptide bond with their BoNT prototype. The sole exception is LC/F5 which has been demonstrated to cleave the bond L 54 -E 55 of VAMP-2 [11], whereas all other analyzed F subtypes (F1, F2, F4, F6, and F7) cleave the Q 58 -K 59 bond [15][16][17][18][19]. The peculiar cleavage activity of BoNT/F5 is explainable by the very high sequence diversity (about 53 %) of LC/F5 from all other LC/Fs (Table S1).…”