Expression of the
            <i>Clostridium botulinum</i>
            A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Lin, Guo; Tepp, William H.; Pier, Christina L.; Jacobson, Mark J.; Johnson, Eric A.

doi:10.1128/aem.01882-09

Cited by 37 publications

(46 citation statements)

References 38 publications

(41 reference statements)

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“…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.)…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

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Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

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“…Culture proteins were concentrated by acid precipitation as previously described (11)(12)(13)57). Briefly, following incubation of C. botulinum for 96 h at 37°C, the cultures were cooled for 60 min on ice, and the pH of the total culture was gradually lowered to 3.5 by slow addition of 1.5 M H 2 SO 4 with gentle stirring.…”

Section: Biosafetymentioning

confidence: 99%

“…Within serotype A, five subtypes of BoNT/A (A1 to A5) have been identified by nucleotide sequence analyses (8-11), and their 150-kDa neurotoxins have been purified (8,(11)(12)(13)(14)(15). Recently, two additional bont/a genes have been reported from distinct dualBoNT-producing strains.…”

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confidence: 99%

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Lin

Tepp

Bradshaw

et al. 2015

Appl Environ Microbiol

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Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. Botulinum neurotoxins (BoNTs) are produced by anaerobic, Gram-positive, spore-forming bacteria, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum. BoNTs are the most poisonous protein toxins known in nature, and they are classified as tier 1 category A select agents due to their potential use as bioterrorism agents (1). Purified BoNT is a protein with a molecular mass of ca. 150 kDa, consisting in its active form of a heavy chain (Hc) (ϳ100 kDa) and a light chain (Lc) (ϳ50 kDa) linked by a disulfide bond and noncovalent molecular interactions (2). The heavy chain contains two functional domains, a receptor binding domain and a translocation domain (3, 4), while the light chain is responsible for catalytic activity on SNARE substrates in the neuronal cell cytosol. BoNTs are distinguished immunologically into seven serotypes designated A to G based on toxin neutralization using homologous antitoxins (5). Recently, the existence of an eighth serotype, H, in a dual-toxin-producing strain of C. botulinum has been reported (5).Of the seven established serotypes, BoNT/A is of particular interest since it is the most potent BoNT, exhibits longer duration in humans than other serotypes, and is also implicated in most cases of human botulism in the continental United States (6, 7). Within serotype A, five subtypes of BoNT/A (A1 to A5) h...

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“…When BoNT and NTNHA are expressed within an ha -orfX + cluster, only the M-PTC has been purified. Eventually, minor amounts of OrfX proteins have been identified in association with the M-PTC or have been observed in crude toxin preparations (Lin et al 2010). So far the function of the OrfX proteins remains unclear.…”

Section: Complexity Of Botulinum Neurotoxinsmentioning

confidence: 99%

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

Dorner

Schulz

Kull

et al. 2012

Current Topics in Microbiology and Immunology

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The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.

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Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

Cited by 37 publications

References 38 publications

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

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

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

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