There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.
Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.
Clostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.
Chromosome and plasmid sequences of several C. botulinum strains representing A, B, E and F serotypes and a C. butyricum type E strain were compared to examine their genomic organization, or synteny, and the location of the botulinum toxin complex genes. These comparisons identified synteny among proteolytic (Group I) strains or nonproteolytic (Group II) strains but not between the two Groups. The bont complex genes within the strains examined were not randomly located but found within three regions of the chromosome or in two specific sites within plasmids. A comparison of sequences from a Bf strain revealed homology to the plasmid pCLJ with similar locations for the bont/bv b genes but with the bont/a4 gene replaced by the bont/f gene. An analysis of the toxin cluster genes showed that many recombination events have occurred, including several events within the ntnh gene. One such recombination event resulted in the integration of the bont/a1 gene into the serotype toxin B ha cluster, resulting in a successful lineage commonly associated with food borne botulism outbreaks. In C. botulinum type E and C. butyricum type E strains the location of the bont/e gene cluster appears to be the result of insertion events that split a rarA, recombination-associated gene, independently at the same location in both species.
The analysis of the genomic sequences representing different strains reveals the presence of insertion sequence (IS) elements and other transposon-associated proteins such as recombinases that could facilitate the horizontal transfer of the bonts; these events, in addition to recombination among the toxin complex genes, have led to the lineages observed today within the neurotoxin-producing clostridia.
A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences—specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.
Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A–G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression.
Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid.
Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum.
Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.
Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs) that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G) and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains.
In the present study, we designed and evaluated the performances of a resequencing microarray (RMA), the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components.
This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays.
The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.
Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex.
An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.
The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.
Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.
Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).
These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.
Botulism; Mass spectrometry; Genomics; Whole genome sequencing
Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.
A polymerase chain reaction method was developed for the specific detection of the botulinum neurotoxin (BoNT) gene of Clostridium botulinum. Degenerate oligonucleotide primers, designed from the nucleotide sequence of the heavy chain of the BoNT gene, amplified a specific fragment of approximately 1.1 kb from strains of C. botulinum toxin types A, B, E, F, and G and neurotoxin-producing strains of Clostridium barati and Clostridium butyricum, but no fragment was obtained from nontoxigenic strains. The fragments amplified from several strains of C. botulinum types B, E, and F were cloned in Escherichia coli and their nucleotide sequences were determined. Sequences within this region were used to design oligonucleotide probes specific for BoNT type B (BoNT/B), BoNT/E, and BoNT/F genes. An additional probe was designed for the detection of the BoNT/F gene of C. barati, which differed in sequence from BoNT/F genes of both proteolytic and nonproteolytic strains of C. botulinum.
Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA− Orfx+ A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA− Orfx+ A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.
Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active di-chain form of the toxin. Nicking of the single chain BoNT/E to the di-chain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 °C, the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.
botulinum neurotoxin; botulism; endopeptidase; SNAP-25; light chain
Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.
Intoxication with the potent botulinum neurotoxin (BoNT) gives rise to the serious paralytic illness botulism. BoNT is part of a complex that consists of the neurotoxin and several associated components, all encoded by the bont gene cluster. This gene cluster has likely been subjected to horizontal gene transfer between different groups of clostridia, which has given rise to the genetically diverse species Clostridium botulinum. C. botulinum is divided into four physiological groups (I–IV), where group I and II cause disease in humans and group III in animals. Analysis of the genomes of group I, II and III has revealed that toxin genes, including the bont cluster, often are plasmid-borne. The genomes analyzed from group III contain an unusually high number of plasmids carrying different toxin genes. Some of these genes are also found in other Clostridium species and some have moved between different plasmids within the same physiological group. This indicates that horizontal transfer of toxin genes is taking place within and between species of Clostridium. The abundance of mobile elements, especially in genomes of group III, is likely connected to accelerated genome plasticity and gene transfer events.
IS element; botulinum neurotoxin; C2 toxin; Clostridium novyi sensu lato; Clostridium botulinum group III; horizontal gene transfer
A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.
Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.
The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.
botulinum neurotoxin; C-D mosaic; botulism; phosphatidylethanolamine; membrane recognition
Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.
Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.
Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.
C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor∶recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.
This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.
Clostridium botulinum, an important pathogen of humans and animals, produces botulinum neurotoxin (BoNT), the most poisonous toxin known. We have determined by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations that the genes encoding BoNTs in strains Loch Maree (subtype A3) and 657Ba (type B and subtype A4) are located on large (~280 kb) plasmids. This is the first demonstration of plasmid-borne neurotoxin genes in Clostridium botulinum serotypes A and B. The finding of BoNT type A and B genes on extrachromosomal elements has important implications for the evolution of neurotoxigenicity in clostridia including the origin, expression, and lateral transfer of botulinum neurotoxin genes.
Clostridium botulinum; botulinum neurotoxin (BoNT); pulsed-field gel electrophoresis (PFGE); Southern hybridization
Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont/B subtype-related.
PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from ∼55 kb to ∼245 kb.
The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.
Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.
The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.
Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.
Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.