Purification, Modeling, and Analysis of Botulinum Neurotoxin Subtype A5 (BoNT/A5) from Clostridium botulinum Strain A661222

Abstract

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most potent neurotoxin known. BoNTs are characterized as category A select agents because of their potential as a bioterrorism threat as listed by the Centers for Disease Control and Prevention (1). BoNTs can be immunologically distinguished by homologous antitoxins into seven primary serotypes, designated A to G. Among these serotype distinctions, there is considerable genetic variation, as demonstrated by the recognition of at least 24 subtypes (3,8,11,17). These subtypes have been distinguished based on their degree of genetic variation, with subtypes having a minimum of 2.6% divergence at the amino acid level (3), but except for BoNT subtypes A1 (BoNT/A1) and -A2, they have not been purified and analyzed at the protein level, which is important to delineate functional differences between the subtypes (15). The purification and characterization of the biochemical, toxicological, and molecular mechanisms of the subtype toxins of various serotypes will provide valuable information as to their biochemical, immunological, and cell biology properties.Recently, a new subtype of BoNT/A was identified and named "BoNT/A5"; there are five strains known to possess the gene encoding BoNT/A5 (3,8). Among these five strains, four of them have neurotoxin sequences that are identical, and the fifth strain has a neurotoxin sequence that is 99.8% identical to the others at the amino acid level. The subtype features both a high degree of similarity to BoNT/A1 and a hemagglutinin (HA)-type gene cluster which is present in only BoNT/A1 clusters and none of the other BoNT/A subtypes. The Eric A. Johnson (E.A.J.) laboratory identified an additional A5 strain of C. botulinum, A661222, which possessed a gene for BoNT/A5 identical to that of strain IBCA94-0216 (8).To investigate the BoNT/A5 of this strain, the neurotoxin gene and its associated genes were completely sequenced and analyzed at both the nucleotide and amino acid level. Thirty-six amino acid differences were observed between BoNT/A1 and BoNT/A5, with most of them in the h...

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

“…The specific toxicities of BoNT/A2, -3, and -5 in mice have previously been reported and differ only marginally from that observed for BoNT/A1 (23,(32)(33)(34). In order to determine the specific activity of BoNT/A4, mice were injected intraperitoneally (i.p.)…”

“…The 150-kDa holotoxins of BoNT/A1, -2, -3, and -5 were purified from C. botulinum strains Hall A hyper, Kyoto-F, CDC A3, and A661222 as previously described (23,(32)(33)(34). The purified toxins were stored in 40% glycerol-phosphate-buffered saline at Ϫ20°C until use.…”

Subtyping Botulinum Neurotoxins by Sequential Multiple Endoproteases In-Gel Digestion Coupled with Mass Spectrometry

“…[4][5][6] For example, gene sequence analysis has identified five BoNT/A subtypes (A1 through A5). [7][8][9] Whereas the sequence similarity among different serotypes is relatively low, the subtypes within a BoNT serotype generally exhibit high sequence similarity. The BoNTs are futher divided into toxin variants that are based on minor sequence differences within a toxin subtype derived from different bacterial strains.…”

“…The subtypes of BoNT serotypes often show a high level of sequence similarity with as little as 2.6% sequence difference and the toxin variants can vary by as little as a single amino acid. 9 Thus, identification of the toxin subtype/variant, which can be a critical factor in responding to and preventing public health emergencies, requires high sequence coverage to expose subtype/variant-specific amino acid variable sites and provide confident discrimination. and may prove useful to differentiate other toxins or human pathogens to aid public health tracking and disease prevention.…”

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

“…For the BoNT/A serotype, genetic analysis has identified five subtypes including A1 to A5 (Arndt, Jacobson et al 2006;Carter, Paul et al 2009;Jacobson, Lin et al 2011). All data described above were obtained using the toxin subtype of BoNT/A1.…”