Genetic Homogeneity of
            <i>Clostridium botulinum</i>
            Type A1 Strains with Unique Toxin Gene Clusters

Raphael, Brian; Lúquez, Carolina; McCroskey, L M; Joseph, Lavin A.; Jacobson, Mark J.; Johnson, Eric A.; Maslanka, Susan E.; Andreadis, Joanne D.

doi:10.1128/aem.00260-08

Cited by 41 publications

(59 citation statements)

References 18 publications

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“…Following our optimized procedure ( Figure S2), a pool of MS-detected peptides generated from the three sets of multi-enzyme, sequential in-gel digestions of the toxin containing gel bands confirmed that the target toxin was serotype A of BoNT (Table S2). The detected 98.6% sequence coverage determined by the 1082 unique peptides enabled identification of this toxin as the subtype BoNT/A1 (GeneBank accession no: ABY56330), consistent with the result of DNA sequencing 23 . Figure 3 shows the sequence alignment of the identified toxin and two homologues of the BoNT/A1 toxin variants (Accession no: ABZ80564 and YP_001383190).…”

Section: Subtyping Of a Bont-contaminated Carrot Juice Samplesupporting

confidence: 72%

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

Wang¹,

Baudys²,

Rees³

et al. 2012

Anal. Chem.

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Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A through G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. But with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.

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Section: Subtyping Of a Bont-contaminated Carrot Juice Samplesupporting

confidence: 72%

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

Wang¹,

Baudys²,

Rees³

et al. 2012

Anal. Chem.

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“…As shown in previous reports, BoNT/A5 and the majority of BoNT/A1 genes are found in ha clusters, while BoNT/A2, BoNT/ A3, and BoNT/A4 genes are in orfX clusters; however, in some strains, the A1 toxin gene is found in an orfX cluster (9,(29)(30)(31)(32)(33)(34). Many strains that produce BoNT/A1 also carry a silent BoNT/B gene (e.g., NCTC 2916) and have both types of clusters, an haassociated bont/b (silent) gene cluster and an orfX-associated bont/a1 cluster.…”

mentioning

confidence: 91%

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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“…In these genomic locations, these genes are associated with toxin accessory genes and form toxin gene clusters. Two main toxin gene clusters, ha and orfX, are known (13)(14)(15)(16).…”

mentioning

confidence: 99%

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

Kenri

Sekizuka

Yamamoto

et al. 2014

Appl Environ Microbiol

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Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/ A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed singlenucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.

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Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters

Cited by 41 publications

References 18 publications

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

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

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

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

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