Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray

Raphael, Brian; Joseph, Lavin A.; McCroskey, L M; Lúquez, Carolina; Maslanka, Susan E.

doi:10.1016/j.mcp.2009.12.003

Cited by 27 publications

(31 citation statements)

References 27 publications

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“…Briefly, the microarray featured 225 probes representing selected regions from various complete C. botulinum group I genome sequences, the bont/A to -G genes, and plasmid-specific regions. Microarray spotting was performed by ArrayIt (Sunnyvale, CA), and hybridizations were carried out as previously described (7).…”

Section: Methodsmentioning

confidence: 99%

Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

Raphael

Bradshaw

Kalb

et al. 2014

Appl Environ Microbiol

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bBotulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/ F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

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Section: Methodsmentioning

confidence: 99%

Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

Raphael

Bradshaw

Kalb

et al. 2014

Appl Environ Microbiol

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“…Genomic sequence data also provided the opportunity to develop microarrays using specially designed oligonucleotides that could quickly differentiate strains based upon the presence or absence of specific genes [106][107][108]87,18,9,124,134,98]. Microarrays have been designed to identify specific C. botulinum Groups or BoNT serotypes and provide information about toxin cluster genes as well as other genes of interest.…”

Section: Microarrays and Pcrmentioning

confidence: 99%

Historical and current perspectives on Clostridium botulinum diversity

Smith

Hill

Raphael

2015

Research in Microbiology

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130

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“…The diversity and variations of neurotoxin gene clusters ( Fig. 1) suggest that horizontal gene transfer recombination has occurred during evolution (29,35,36).…”

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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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Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray

Cited by 27 publications

References 27 publications

Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

Historical and current perspectives on Clostridium botulinum diversity

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

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