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Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

ABSTRACT: Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

SUBMITTER: Lin G

PROVIDER: S-EPMC2798633 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

Lin Guangyun G Tepp William H WH Pier Christina L CL Jacobson Mark J MJ Johnson Eric A EA

Applied and environmental microbiology 20091113 1

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Po ...[more]

PMID: 19915042

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Project description:Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

Dataset Information

Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

Publications

Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

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