Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins

Abstract

Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs) to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH), and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC), but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs) which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex.

Keywords: botulinum neurotoxin; botulism; protein toxin; proteomics; toxin complex.

Figure 1

MS/MS of unique peptides TNDKDLIGTLLIEAGSSGSIIQPR from HA-70 (A); WLINPVSDTDETYTITNLR from HA-33 (B); and YLSYDNFGFISLDSLSNR from HA-17 (C) of BoNT/A1 (botulinum neurotoxin type A1).

Figure 2

MS/MS of unique peptides NKFNLYVINEDIEKR from Orf-X1 (A); FTQIFSYILLNETSK from Orf-X2 (B); and TQSDNPEDPAIIVISSYK from Orf-X3 of BoNT/E3 (C). Y-ions are depicted in blue, b-ions are depicted in red, and the precursor ions are depicted in green.

Figure 3

Sequence alignment of the NTNH proteins associated with BoNT/A1(B). The sequence coverage of NTNH-A is depicted in bold and green, and the sequence coverage of NTNH-B is depicted in bold and blue. The MS/MS of the underlined peptides are in Figure 4, and the sections largely responsible for binding to HA-70 in BoNT/A HA+ progenitor toxin complexes are highlighted.

Figure 4

MS/MS of peptide YDQFYVDPALELIK from NTNH associated with the bont/A1 cluster (A), and peptide YDEFYIDPAIELIK from NTNH associated with the bont/(b) cluster (B).