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

Abstract

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins; botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5 while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies, and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity towards SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies, but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

Impact of Clostridium botulinum genomic diversity on food safety

“…Hybrids of type C and type D neurotoxin (type C/D or type D/C) are formed more frequently than type C or type D neurotoxin [30]. A highly novel neurotoxin (type FA (Figure 1), also known as type H and type HA) that contains regions of similarity to type A neurotoxin and type F neurotoxin has been recently identified and is being characterised [31, 32, 33, 34, 35, 36]. Hybrid neurotoxin subtypes have been identified, e.g.…”

Clinical differences between botulinum neurotoxin type A and B

“…BoNT is produced by Clostridium botulinum and includes eight antigenically distinct toxins, labeled A, B, C, D, E, F, G and H, although the existence of the H, most recently identified Dover et al, 2014) has been questioned (Johnson, 2014;Kalb et al, 2015).…”

Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach

“…BoNTs produced by anaerobic Clostridium botulinum are the cause of botulism, a life-threatening neuroparalytic disease. There are currently seven known serotypes of BoNTs, designated as types A to G. More recently, the eighth serotype, type H, has been identified (3), and it is believed that BoNT/H is a hybrid of known serotypes of A and F (4, 5). Serotypes A, B, E, and occasionally F have been shown to cause human botulism, and among them, type A is the most potent and has the longest paralysis effect in vivo (up to 6 months) (1, 2).…”