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

Wang, Dongxia; Baudys, Jakub; Rees, Jon C.; Marshall, Kristin M.; Kalb, Suzanne R.; Parks, Bryan A.; Nowaczyk, Louis; Pirkle, James L.; Barr, John R.

doi:10.1021/ac3006439

Cited by 13 publications

(17 citation statements)

References 30 publications

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“…For instance, the translocation domain of E10 is highly homologous to that found in all variants except E9 and E11, while large portions of its catalytic and receptor-binding domains are identical to those of E11. Such patterns suggest the occurrence of recombination events and have been previously reported for other bont genes, including bont/E variants, and toxin gene clusters (2,9,15,28). The frequency of nucleotide substitutions observed within variants E10 (1 to 7 nucleotides) and E3 (1 or 2 nucleotides) is similar to that reported for variant B4 (1 to 5 nucleotides), a toxin variant generally associated with nonproteolytic C. botulinum (29).…”

Section: Discussionsupporting

confidence: 83%

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

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Lambert

Mabon

et al. 2014

Appl Environ Microbiol

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We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay. B otulism, a rare and severe disease characterized by a descending flaccid paralysis, is caused by botulinum neurotoxin (BoNT), the most potent toxin known. BoNT is produced by phylogenetically distinct anaerobic spore-forming bacteria grouped under the taxonomic designation of Clostridium botulinum. Rare botulinogenic strains of related clostridia, such as C. baratii, C. butyricum, and C. argentinense, have also been observed (1, 2). Seven serologically distinct BoNTs (A to G) can be distinguished based on neutralization of toxicity with specific antisera. Recently, a strain of C. botulinum producing botulinum neurotoxin type B (BoNT/B) and another BoNT that is not neutralized by antitoxins to BoNTs A to G has been isolated from a case of infant botulism (3). It has been proposed that this novel neurotoxin is an eighth serotype, BoNT/H (3,4). The botulinum neurotoxins are comprised of three structural domains (translocation [H N ], receptor binding [H C ], and catalytic [LC]). These toxins target different SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins in the neuromuscular junction to block neurotransmitter release (5,6).Heterogeneity within the BoNTs has led to a classification of BoNTs into subtypes. Within a toxin serotype, differences in amino acid sequences can range from 0.9% to 25% (2, 7). Hill and Smith (8) point out that the newer subtypes have been defined based on their DNA sequence and propose the use of the term "subtype/genetic variant" to avoid confusion with the historical use of "subtype," utilized to designate immunological or enzymatic differences among neurotoxins. Two approaches have been used to define new BoNT variants. The first uses a cutoff value of 2.5% difference in amino acid composition (9-11), whereas the second relies on a phylogenetic approach in which variants correspond to clades formed by the clustering of bont sequences (1, 7, 12) Based on these methods, bont/A1 to bont/A5, bont/B1 to bont/ B7, bont/E1 to bont/E9, and bont/F1 to bont/F7 gene variants have been described (1,2,8,10,(12)(13)(14)(15)(16)(17).In Canada, C. botulinum type E is the predominant BoNT serotype associated with food-borne botulism, accounting for 86.2% of all laboratory-confirmed food-borne botulism outbreaks reported between 1985 and 2005 (18). Although variant types E1, E3, and E7 have been identified in t...

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Section: Discussionsupporting

confidence: 83%

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

Weedmark

Lambert

Mabon

et al. 2014

Appl Environ Microbiol

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“…Furthermore, Endopep-MS detects active toxin down to serotype level. Also, it has been demonstrated that the subtype can be determined by a subsequent proteomics approach applied on the antibody captured toxin after the Endopep-MS procedure [22,26,40,41]. There are several successful examples of the use of Endopep-MS for the detection of BoNTs in different matrices [42,38].…”

Section: Introductionmentioning

confidence: 99%

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum

et al. 2014

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Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform an in-house validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, E, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C, D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a good alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, speed, and that it does not require experimental animals.

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“…Using antibodies to the toxin, the toxin has been isolated from food [43–45] or bacterial culture [44, 46]. Through this process, both BoNT/A [43–45] and BoNT/B [44, 46] were reported to be detected even in the presence of complex matrices. The sequence coverage on the neurotoxin using this process was reported as between 65 and 98%.…”

Section: Bont Serotype Detection In Complex Matricesmentioning

confidence: 99%

“…In 2012, Wang et al reported on a new technique to improve sequence coverage of BoNT proteins [45]. This work consisted of separation of the protein complex components by SDS-PAGE followed by digestion of each gel band with multiple enzymes and sequential in-gel digestion.…”

Section: Differentiation Of Bont At the Subtype/toxin Variant Levelmentioning

confidence: 99%

“…Peptides from the gel band containing the neurotoxin were then separated by LC and analyzed by MS/MS followed by database searching. This analysis yielded sequence coverage of 90% and greater for all serotypes of BoNT [45]. Furthermore, this technique was used to identify the toxin present in a toxin-contaminated carrot juice sample associated with a complex botulism outbreak.…”

Section: Differentiation Of Bont At the Subtype/toxin Variant Levelmentioning

confidence: 99%

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Mass spectrometric identification and differentiation of botulinum neurotoxins through toxin proteomics

Kalb

Barr

2013

Reviews in Analytical Chemistry

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Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence and immunogenic properties, and some subtypes are further differentiated into toxin variants. Toxin characterization is important as different types of BoNT can respond differently to medical countermeasures for botulism, and characterization of the toxin can aid in epidemiologic and forensic investigations. Proteomic techniques have been established to determine the serotype, subtype, or toxin variant of BoNT. These techniques involve digestion of the toxin into peptides, tandem mass spectrometric (MS/MS) analysis of the peptides, and database searching to identify the BoNT protein. These techniques demonstrate the capability to detect BoNT and its neurotoxin-associated proteins, and differentiate the toxin from other toxins which are up to 99.9% identical in some cases. This differentiation can be accomplished from toxins present in a complex matrix such as stool, food, or bacterial cultures and no DNA is required.

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

Cited by 13 publications

References 30 publications

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum

Mass spectrometric identification and differentiation of botulinum neurotoxins through toxin proteomics

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