Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

Abstract

ᰔBotulinum neurotoxin, the most poisonous substance known and a potential biothreat agent (1), causes human and animal botulism worldwide (4,9). This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinincontaining (haϩ) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7, 10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3,8).Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2). The California infant strain, designated IBCA94-0216, was characterized by sequencing the toxin complex genes and their flanking regions (GenBank accession number FJ959094). Sequence analysis confirmed that the bont/A5 is located within a hemagglutinin complex (haϩ) that contains the genes ha70, ha17, ha33, botR, ntnh, and bont/A5 (Fig. 1). This type of toxin cluster is commonly associated with strains bont/A1 and bont/B but is not found in strains bont/A2, bont/A3, and bont/A4 (7, 10). The IBCA94-0216 neurotoxin gene cluster has an unexpected 76-bp deletion between the genes ha33 and botR. This is the first example of such a deletion.Downstream from bont/A5 are two copies of transposases belonging to the is3 family, a structure identical to the downstream flanking sequence of the type A1 Hall neurotoxin gene cluster ( Fig. 1) (10). Interestingly, 2,041 bp downstream from the bont/A5 gene and immediately following the second is3 is a partial bont/B gene that is only 65% of a full-length bont/B (6). The partial bont/B gene lacks the nucleotide sequence coding for the light chain of the toxin and begins at nucleotide 1362 of the 3,876-nucleotide full-length toxin gene. Comparisons of this region (1,362 to 3,876 nucleotides) revealed it is most similar to the rare bont/B3 subtype (5). The entire IBCA94-0216 bont/A/B cluster is flanked by a 5Ј flagellin gene and a 3Ј transposase, as is found in the silent type B gene cluster of strain NCTC2916 (7).Nucleic acid pairwise identities (using the Kimura two-parameter method in MEGA4 software) of the toxin cluster genes show that the IBCA94-0216 A5 neurotoxin and ntnh genes are most similar to the type A1 Hall neurotoxin and ntnh genes, respectively, while the hemagglutinin genes are most similar to the hemagglutinin genes of type B strains. This mosaic gene arrangement and the presence of the partial type B neurotoxin gene lead us to speculate that some form of genetic rearrangement occurred between type A and type B neurotoxin gene clusters in the c...

Further Characterization of Proteolytic Clostridium botulinum Type A5 Reveals that Neurotoxin Formation Is Unaffected by Loss of the cntR ( botR ) Promoter Sigma Factor Binding Site

“…1). Recently, a very similar arrangement in a type A5 infant botulism strain (IBCA94-0216) was reported by Dover et al (4). The two type A5 neurotoxin clusters share 99.7% nucleotide sequence identity between the 3Ј end of the cntE (ha70) gene and the 3Ј end of the type B3 neurotoxin-encoding gene.…”

“…Of 38 other strains of proteolytic C. botulinum also possessing the ha ϩ form of the neurotoxin gene cluster (8), selected as representing a wide range of diseases caused, geographical distribution and type of neurotoxin(s) formed (3), only the type B toxin-forming strain 2345 shared the same deletion, implying that this modification is quite rare. Dover et al (4) suggested that the A5 neurotoxin gene, together with its cntB (ntnh) gene, may have been inserted into a type B neurotoxin cluster. Since microarray analysis showed that strain 2345 clustered with Clostridium sporogenes (3), it is tempting to postulate that a strain of C. sporogenes acquired a type B neurotoxin cluster by horizontal transfer to give a strain similar to strain 2345, which, following a further round of recombination at this locus, gave rise to the type A5 neurotoxin cluster now described.…”

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

“…[4][5][6] For example, gene sequence analysis has identified five BoNT/A subtypes (A1 through A5). [7][8][9] Whereas the sequence similarity among different serotypes is relatively low, the subtypes within a BoNT serotype generally exhibit high sequence similarity. The BoNTs are futher divided into toxin variants that are based on minor sequence differences within a toxin subtype derived from different bacterial strains.…”

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

“…Group III encompasses serotype C and D strains, while group IV is only composed of serotype G strains (4,21). Differences in the BoNT gene nucleotide sequences lead to the definition of five subtypes (A1 to A5) for type A, five (proteolytic B1 to B3, bivalent B, and nonproteolytic B) for type B, four (E1 to E3 and E6) for type E, and seven (F1 to F7) for type F (2,3,6,23). Two subtypes from BoNT/E-producing Clostridium butyricum strains (subtypes E4 and E5) have been described (3).…”