TY - JOUR
AU - Baumstark, Barbara, R
AB - Abstract Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain. Clostridium botulinum, Neurotoxin gene sequence, Polymerase chain reaction 1 Introduction Clostridium botulinum toxins are responsible for the disease botulism in humans and animals. There are seven antigenically distinct toxin types (A–G), four of which primarily affect humans. Each of the botulinum toxins, consisting of a light chain and a heavy chain, is encoded by a single open reading frame (ORF). The light chain, which is about one third of the polypeptide, is encoded first, followed by the heavy chain. The two chains are cleaved by a proteolytic event and covalently linked by a disulfide bond to form the active botulinum neurotoxin. Due to the severity of botulism intoxication, several methods have been developed for the identification of botulinum neurotoxin in contaminated food samples. The standard method accepted by the Food and Drug Administration (FDA) is the mouse bioassay, with one minimum lethal dose (MLD) being defined as the lowest dose sufficient to intoxicate and kill a pair of mice. Although most botulinum strains express a single antigenically distinct toxin, a few isolates have been shown to produce more than one toxin type. To date, strains with three different combinations of toxin genes have been reported and designated Ab (4, 5, 16) [1–3], Af (6, 1) [4,5], Ba (7, 9) [6,7] and Bf (10) [8], where the smaller case letter indicates the toxin with lesser activity. Franciosa et al. [4] reported that over 50% of strains designated as type A by the mouse bioassay encode sequences for both type A and type B genes, as determined by polymerase chain reaction (PCR) amplification. Because no type B toxin activity was detected in these strains, it was concluded that they contained a defective, or cryptic, copy of the type B gene. These strains were designated type A(B) to indicate the presence of gene sequences corresponding to the type B toxin without any corresponding type B-specific toxicity. The cryptic type B gene of one type A(B) strain (strain 667) was reported to contain multiple mutations within the toxin ORF (13) [9]. A limited number of strains were isolated and found to express significant levels of type A-specific toxin and extremely low levels (less than 0.1% wild-type values) of active type B-specific toxin. These strains have been designated type Ab(4) [1]. In this report, we have characterized four isolates of C. botulinum which were designated as type A by the mouse bioassay but which were shown to have both type A and type B gene sequences by PCR diagnostic assays. In addition, we have characterized a fifth isolate that expresses both type A and type B toxicity as determined by the mouse bioassay. 2 Materials and methods 2.1 Bacterial strains The C. botulinum strains (Table 1) used in this study were provided by Charles Hatheway (Centers for Disease Control and Prevention). Strain BGB, a proteolytic type B toxin producer isolated from green beans, was obtained from the FDA Southeast Regional Laboratory culture collection. Strain A73, a type A toxin producer, was provided by Haim Solomon, FDA-CFSAN (Center for Food Safety and Applied Nutrition). 1 Source and geographic locations of the C. botulinum strains Strain Type Geographic location Year isolated Source 519 A(B) Alaska 1976 stool (salmon eggs) 588 Ab Ohio 1976 stool (carrots) 593 A(B) Georgia 1976 dog feces 667 A(B) Wisconsin 1976 stool 13280 A(B) Colorado 1972 peppers 1436 AB Utah 1977 stool (unknown) 6440 B Arizona 1987 infant Strain Type Geographic location Year isolated Source 519 A(B) Alaska 1976 stool (salmon eggs) 588 Ab Ohio 1976 stool (carrots) 593 A(B) Georgia 1976 dog feces 667 A(B) Wisconsin 1976 stool 13280 A(B) Colorado 1972 peppers 1436 AB Utah 1977 stool (unknown) 6440 B Arizona 1987 infant Open in new tab 1 Source and geographic locations of the C. botulinum strains Strain Type Geographic location Year isolated Source 519 A(B) Alaska 1976 stool (salmon eggs) 588 Ab Ohio 1976 stool (carrots) 593 A(B) Georgia 1976 dog feces 667 A(B) Wisconsin 1976 stool 13280 A(B) Colorado 1972 peppers 1436 AB Utah 1977 stool (unknown) 6440 B Arizona 1987 infant Strain Type Geographic location Year isolated Source 519 A(B) Alaska 1976 stool (salmon eggs) 588 Ab Ohio 1976 stool (carrots) 593 A(B) Georgia 1976 dog feces 667 A(B) Wisconsin 1976 stool 13280 A(B) Colorado 1972 peppers 1436 AB Utah 1977 stool (unknown) 6440 B Arizona 1987 infant Open in new tab 2.2 Enzyme-linked immunosorbent assay (ELISA) analysis for type A and type B toxins Determination of toxin levels by ELISA was conducted following the procedure of Ferreira et al. [10]. Briefly, goat anti-A or anti-B antibodies were fixed in carbonate buffer to microtiter plate wells overnight at 4°C. The plate was washed and blocked with 1% bovine serum albumin (BSA). Each culture broth was placed in type A and type B wells and incubated at 25°C for 1 h. Rabbit anti-A or anti-B antibodies were used followed by an anti-rabbit alkaline phosphatase conjugate to detect toxin captured by the goat antibodies. After washing, p-nitrophenyl phosphate substrate was added, the reaction was then incubated at 35°C for 1 h, and the absorbance was measured at 410 nm. 2.3 Amplification of overlapping toxin type B gene fragments C. botulinum genomic DNA was isolated as described previously [11]. The Roche Molecular Biochemicals (Indianapolis, IN, USA) Expand kit was used to amplify overlapping type B toxin gene fragments (Fig. 1) from purified genomic DNA for each of the five A(B) and the wild-type strain BGB as specified by the manufacturer. The thermal cycling program was used as follows: after a denaturation step at 92°C for 2 min, three cycles of 92°C for 20 s, 50°C for 30 s, and 68°C for 3 min were performed. This was followed by 25 cycles of 92°C for 20 s, 50°C for 30 s, 68°C for 3 min (plus 20 s extra/cycle), and a one-step extension at 68°C for 7 min. 1 Open in new tabDownload slide ELISA analysis of the C. botulinum A(B), Ab, and A/B strains. The ELISA protocol was followed as described in Section 2, using anti-type A antibodies (A) or anti-type B antibodies (B). The results are presented as bar graphs for the 10−1 dilution of the botulinum cultures: 519, 588, 593, 667, 1436, 13280. Type A strain A73 was used as positive control for the type A ELISA and type B strain 6440 was used as positive control for the type B ELISA. 1 Open in new tabDownload slide ELISA analysis of the C. botulinum A(B), Ab, and A/B strains. The ELISA protocol was followed as described in Section 2, using anti-type A antibodies (A) or anti-type B antibodies (B). The results are presented as bar graphs for the 10−1 dilution of the botulinum cultures: 519, 588, 593, 667, 1436, 13280. Type A strain A73 was used as positive control for the type A ELISA and type B strain 6440 was used as positive control for the type B ELISA. 2.4 DNA sequencing and analyses The overlapping DNA amplification fragments of the wild-type and cryptic type B toxin genes were purified using the Qiagen (Valencia, CA, USA) Qiaquick columns for subsequent sequencing. The PCR-based ABI automated sequencing system was used to elucidate the sequences of the DNA amplification products in both directions. The sequencing reactions were performed and the products were purified following the fluorescent dye deoxy protocol and electrophoresed in the 373 DNA Sequencer (PE Biosystems, Foster City, CA, USA). The sequence was then analyzed using the MacDnasis (Hitachi Software) DNA analysis program. 2.5 RNA analyses Total RNA was isolated from C. botulinum cultures grown under anaerobic conditions overnight in 10 ml of TPGY broth (tryptone, 50.0 g l−1; peptone, 5.0 g l−1; dextrose, 4.0 g l−1; yeast extract 20.0 g l−1; sodium thioglycolate, 1.0 g l−1). The cells were washed and resuspended in 1 ml of TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM disodium ethylenediamine tetraacetic acid (EDTA)). 100 μl of the washed cells were then resuspended in 100 μl of lysis buffer containing 1.0 mg lysozyme ml−1 TE buffer. The RNA purification procedure was followed using the RNeasy RNA purification kit (Qiagen, Inc., Valencia, CA, USA) following the manufacturer's instructions. 3 μg of total RNA were used for Northern hybridization as previously described [12]. 3 Results and discussion 3.1 Characterization of the type A botulinum strains containing type B neurotoxin sequences All six C. botulinum strains (Table 1) examined exhibited wild-type levels of type A toxicity as determined by the mouse bioassay and were positive by PCR using either type A or type B neurotoxin gene-specific primers. Four isolates (strains 519, 593, 667, and 13280) were designated type A(B) because they exhibited no detectable type B toxicity by the mouse bioassay. The fifth isolate (strain 1436) exhibited a type B-specific toxic response in mice of 40 000 MLD, a value similar to wild-type strains containing only type B-specific toxin. Strain 1436 was designated as type A/B. The sixth isolate (strain 588) produced a very low type B-specific toxicity value by the mouse bioassay (25 MLD) and was classified as type Ab. 3.2 Expression analysis of type B botulinum neurotoxin by ELISA To examine the protein levels of the neurotoxin types A and B in the botulinum isolates, immunoreactivity was monitored by capture ELISA using anti-type A and anti-type B antibodies. ELISA analyses were carried on serially diluted botulinum cultures at each step (10−1, 10−2, 10−3, and 10−4). Significant levels of type A toxin were detected in all of the isolates tested at all dilutions (data not shown). The data for the type B neurotoxin from the undiluted cultures and the 10−4 dilution are shown in Fig. 1. ELISA titers similar to or exceeding that of a control type B strain (strain 6440) were observed in culture fluid isolated from strain A/B 1436 and the minimally toxic type B strain Ab 588 (Fig. 1). No type B neurotoxin protein was discerned in A(B) 519, 593, 667, and 13280, the four strains which did not exhibit any detectable type B toxicity in the mouse bioassay (Fig. 1). The C. botulinum type A (A73) and type B (6440) strains were used as corresponding controls for the type A and type B ELISA analyses (data not shown). 3.3 Sequence analyses of the type B neurotoxin gene The entire ORF for the type B toxin for each of the six isolates was amplified by PCR and subsequent DNA sequence analyses were carried out to determine the molecular basis for the defects in the aberrant type B toxin genes. Control amplification and sequencing reactions were carried out on the proteolytic type B toxin gene of strain BGB. The results of the sequence analysis of the BGB toxin gene yielded an identical DNA sequence to that previously reported by Whelan et al. [12] over the entire 3876 bp ORF. Based on the sequence data on the six newly characterized isolates, we divided the strains into three groups (Fig. 2). Members within each group had identical sequences throughout the ORF. The sequences of the type B toxin genes reported here were more similar to the toxin sequences of the proteolytic type B strain BGB than to that of the non-proteolytic Eklund 17B strain [14]. 2 Open in new tabDownload slide Sequence analysis of type B NT genes in natural isolates. The DNA sequences for the type B neurotoxin genes of the C. botulinum isolates 519, 588, 593, 667, 1436, and 13280 were submitted to GenBank and received the following accession numbers: AF300467 for strain 519; AF300465 for strain 588; AF300466 for strain 593; AF300468 for strain 667; AF295926 for strain 1436; AF300469 for strain 13280. Thin and thick lines indicate sequences coding for the light and heavy chains, respectively. Kilobase pairs are given above the figure. A: Comparison of Group 1 (strain 1436) with BGB. Vertical lines indicate the positions where the Group 1 sequence differs from that of BGB. B: Comparison of Groups 2 and 3 with Group 1 and BGB. Positions in which these groups deviate from both Group 1 and BGB are indicated by solid vertical lines below the midpoint line. Positions where the sequence differs from Group 1 but retains identity to BGB are illustrated by dotted lines above the midpoint line. The sites at which the base pair substitutions result in amino acid changes unique to Group 2 are indicated. Non: nonsense mutation; del: 6 bp deletion; d: 1 bp deletion. 2 Open in new tabDownload slide Sequence analysis of type B NT genes in natural isolates. The DNA sequences for the type B neurotoxin genes of the C. botulinum isolates 519, 588, 593, 667, 1436, and 13280 were submitted to GenBank and received the following accession numbers: AF300467 for strain 519; AF300465 for strain 588; AF300466 for strain 593; AF300468 for strain 667; AF295926 for strain 1436; AF300469 for strain 13280. Thin and thick lines indicate sequences coding for the light and heavy chains, respectively. Kilobase pairs are given above the figure. A: Comparison of Group 1 (strain 1436) with BGB. Vertical lines indicate the positions where the Group 1 sequence differs from that of BGB. B: Comparison of Groups 2 and 3 with Group 1 and BGB. Positions in which these groups deviate from both Group 1 and BGB are indicated by solid vertical lines below the midpoint line. Positions where the sequence differs from Group 1 but retains identity to BGB are illustrated by dotted lines above the midpoint line. The sites at which the base pair substitutions result in amino acid changes unique to Group 2 are indicated. Non: nonsense mutation; del: 6 bp deletion; d: 1 bp deletion. Group 1 was represented by only a single isolate, strain A/B 1436, which exhibited wild-type A and B toxin activity. This isolate differed significantly in the sequence of the type B neurotoxin ORF from those reported earlier [13,14]. Comparative analysis of the type B neurotoxin genes of type A/B strain 1436 and the wild-type BGB strain revealed 75 bp differences within the ORF that result in 33 amino acid changes. Since 1436 and BGB produced active B neurotoxin at comparable levels, the sites at which these substitutions occurred are unlikely to be critical for toxin activity. The type B toxin defective strains 588 and 593 were placed in Group 2, according to their sequence identity. The toxin gene sequences in Group 2 resembled more closely the Group 1 sequence than that of the BGB strain. In all, 61 bp substitutions (relative to BGB) were common to all the three groups. The Group 2 toxin gene sequence shared 25 of 33 of the predicted amino acid substitutions observed for Group 1. The Group 2 sequence contained eight additional base pair substitutions, three of which resulted in alterations at the amino acid level that were not observed in the Group 1 sequence. Because the cryptic B toxin ORF from the strains of Group 2 contained multiple mutations, it was difficult to identify the precise mutation(s) responsible for loss of activity. However, many of the differences between the Group 2 ORF and the two previously published wild-type sequences (the proteolytic BGB strain and the non-proteolytic Eklund 17B strain) were also present in the Group 1 strain (strain 1436), which produced toxin of comparable activity to that of the other wild-type strains. A comparison of Group 1 and Group 2 ORFs revealed three differences that affect the amino acid sequence. Two of the altered amino acids were located in the C-terminal region of the heavy chain, a region that has been implicated in the binding to the neuronal receptor [15]. One of these differences was located at position 1117, where Group 1 and Group 2 code for a proline and a serine, respectively. However, each of these amino acids was represented in one of the previously published wild-type sequences (proline in BGB and serine in the Eklund strain). Therefore, it was unlikely that this difference was critical for function. The second difference was located at amino acid 1182. In this case, the three wild-type strains had unique amino acids at this position, with the BGB, Eklund, and Group 1 strains containing threonine, alanine, and isoleucine, respectively. Group 2 had a methionine at this site. All of these amino acids were non-polar and would not normally be expected to have a great effect on function. The third difference, located near the amino-terminus, resulted in a conservative change from a threonine to a methionine at amino acid 29 that could conceivably affect proteolytic activity of the type B light chain. This alteration did not affect the sequence of the conserved HELIHAGH active site. However, the possibility remained open that the amino acid at this position played an indirect role in maintaining the structural integrity of the catalytic site. Strains in Group 3 (519, 667, and 13280) showed a greater divergence from the BGB strain than those of the other two groups. The Group 3 type B toxin gene contained 16 additional point mutations, a 6 bp deletion at position 985–990 that removed two codons, and a 1 bp deletion at position 2891 that altered the ORF (Fig. 2B). One of the point mutations in Group 3, a G→T transversion at position 382, produced an amber mutation (Fig. 2B). This mutation was predicted to generate a protein fragment of 128 amino acids, corresponding to less than 10% of the normal toxin protein. The severe truncation of the protein resulting from this mutation could account for the absence of type B toxicity and was consistent with the ELISA data for these strains, in which no toxin protein was detected (Fig. 1). The type B sequence for A(B) strain 667 published by Hutson et al. [9] differed from our reported nucleotide sequence for this strain at nine positions. To determine whether the PCR amplification methods used in our analysis may have introduced sequencing artifacts, at least two independently amplified PCR fragments were sequenced from all regions of the ORF, and no sequence anomalies were detected. In addition, PCR fragments corresponding to each region of the toxin gene were amplified from the wild-type type BGB strain in parallel with type 667 and sequenced as controls. At all amplification/sequencing stages, we detected no disparities between our sequence and the sequence previously reported for BGB. Furthermore, we carried out sequence analyses on a second culture of strain 667 (provided by Charles Hatheway, CDC) and confirmed the sequence we obtained with the original isolate. At the present time, we have no explanation for the differences between the sequence reported here for strain 667 and that reported previously [9]. 3.4 Expression analyses of the type B neurotoxin mRNA for strains 519 and 588 The sequence identity in the type B neurotoxin ORF of strains 588 and 593, combined with the distinctly different phenotypes exhibited by these two strains with respect to type B toxin production, as indicated both by the mouse bioassay and the ELISA results (Fig. 1), suggest that these apparent differences were not due to differences in the structural gene itself but to variations in the expression of the gene. A mutation (or mutations) outside the ORF could lead to differences in the efficiency with which this ORF is transcribed, thereby accounting for the differences in protein levels. To determine whether this might be the case, we compared the levels of type B toxin-specific mRNA recovered from strains 588 and 593. Northern analyses of RNA isolated from these strains (Fig. 3) showed two type B-specific transcripts with sizes (4 and 6 kb) corresponding approximately to those reported previously for the type A gene [16], and slightly smaller than those reported for the type B gene [17]. Several poorly resolved bands (≤1.9 kb) were also detected and attributed to degradation. Using optical densitometric analysis of the Northern blots and normalizing for the relative amount of RNA loaded on the gel, strain 588 RNA isolates were found to express type B-specific mRNA sequences at levels 6–7-fold higher than the isolates from strain 593 (Fig. 3) and the two wild-type isolates, strains 1436 and BGB (data not shown). This observation suggested that the mutations in Group 2 did not totally abolish type B toxin activity and that the minimal neurotoxin activity produced by strain 588 resulted from an enhanced expression of a marginally active gene product. Such enhanced expression by strain 588 might be attributable to differences in promoter sequence. Comparative sequence analysis of DNA from strains 588 and 593 in the region containing the proximal promoter revealed no differences, with both strains showing the same sequence as the wild-type strain (Fig. 4). Thus, differences responsible for the phenotypic differences in expression must be localized to a more distal region of the genome, possibly from a transcriptional readthrough from a second more distal promoter that directs expression of the NTNH gene located immediately upstream of the neurotoxin gene [17]. 3 Open in new tabDownload slide Expression analysis of the type B toxin gene in the C. botulinum strains 588 and 593 by Northern hybridization. The nylon membrane-bound RNA from cell cultures was hybridized to a radiolabeled probe spanning most of the type B toxin gene sequence. As described in Section 2, autoradiography was performed after washing the membrane to detect type B neurotoxin-specific mRNA (A and B). A: 1 h exposure. B: 12 h exposure. C: Ethidium bromide-stained agarose gel showing the ribosomal RNA bands to demonstrate equal loading of RNA. 3 Open in new tabDownload slide Expression analysis of the type B toxin gene in the C. botulinum strains 588 and 593 by Northern hybridization. The nylon membrane-bound RNA from cell cultures was hybridized to a radiolabeled probe spanning most of the type B toxin gene sequence. As described in Section 2, autoradiography was performed after washing the membrane to detect type B neurotoxin-specific mRNA (A and B). A: 1 h exposure. B: 12 h exposure. C: Ethidium bromide-stained agarose gel showing the ribosomal RNA bands to demonstrate equal loading of RNA. 4 Open in new tabDownload slide DNA sequence of the promoter region immediately upstream of the type B toxin gene of strains 588 and 593. The ribosome binding site (RBS) and the potential −10 and −35 regions are underlined. The beginning of the ORF is indicated by the arrow. 4 Open in new tabDownload slide DNA sequence of the promoter region immediately upstream of the type B toxin gene of strains 588 and 593. The ribosome binding site (RBS) and the potential −10 and −35 regions are underlined. The beginning of the ORF is indicated by the arrow. In summary, we have sequenced the type B neurotoxin gene in C. botulinum isolates containing both type A and type B neurotoxin genes. Whereas the type A neurotoxin in these strains exhibited protein and activity levels similar to that of wild-type neurotoxin type A, various levels of expression and activity were observed for the type B neurotoxin. Even though these strains were isolated from a wide geographical section across the USA, sequence analysis determined that these strains could be divided into three distinct groups according to their sequence identity. 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Google Scholar PubMed OpenURL Placeholder Text WorldCat © 2004 Federation of European Microbiological Societies
TI - Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences
JF - FEMS Microbiology Letters
DO - 10.1016/S0378-1097(03)00911-X
DA - 2004-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-six-type-a-strains-of-clostridium-botulinum-that-TQySeZ5iny
SP - 159
EP - 164
VL - 231
IS - 2
DP - DeepDyve
ER -