Sequencing the Botulinum Neurotoxin Gene and Related Genes in Clostridium botulinum Type E Strains Reveals orfx3 and a Novel Type E Neurotoxin Subtype

Abstract

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.Clostridium botulinum strains produce one or two of the lethal botulinum neurotoxins (BoNTs) designated A to G according to antigenic properties. These toxins are produced as a progenitor complex consisting of BoNT, hemagglutinin (HA), nontoxic non-HA (NTNH), and probably other uncharacterized components, such as P47 and OrfX (14, 15). The genes encoding these proteins are linked as a cluster and vary in composition and structure among different serotypes and strains. In general, the ntnh and bont genes are located conjointly and arranged in the downstream region of the gene cluster, whereas the upstream regions vary between different types and subtypes of C. botulinum (15).C. botulinum type E predominates in aquatic environments. It differs from the terrestrial toxinotypes of C. botulinum in the arrangement of the neurotoxin gene cluster. Kubota et al. showed that in C. botulinum strain Iwanai, the orfx2, orfx1, and p47 genes are arranged sequentially in the upstream region of the type E neurotoxin gene cluster (19). The partial orfx2 (676 bp) was found in the distal upstream region in the same orientation as orfx1, whereas p47 was located immediately upstream of ntnh and had the opposite orientation to orfx2 and orfx1. The type E neurotoxin-producing Clostridium butyricum strain BL6340 was further shown to be identical to C. botulinum Iwanai in toxin gene arrangement (19). To date, no more sequences of type E neurotoxin gene clusters are available, and the sequences of the complete orfx2 gene and the further upstream region of orfx2 in type E strains are unknown. Type A2 and F strains have orfx2, orfx1, and p47 genes similar to those of type E but with a regulator gene, botR, located ...

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).…”

First Report Worldwide of an Infant Botulism Case Due to Clostridium botulinum Type E

“…Subtype E3 has been identified in strains isolated from clinical and food specimens associated with food-borne botulism outbreaks in the United States and from fish in Germany and Finland (7,11). Two of the most recognized potential sources of BoNTproducing clostridium spores in infant botulism cases are honey and dust (14).…”

“…Based on the amino acid sequences of the neurotoxin, BoNT type E-producing strains of C. botulinum and C. butyricum have been classified into six subtypes (7). Subtypes E1, E2, E3, and E6 include C. botulinum strains, and Acid from:…”

Production of a Neutralizing Mouse-Human Chimeric Antibody against Botulinum Neurotoxin Serotype E

“…In 2007, the first case of infant botulism due to C. botulinum type E was reported in the United States (10). BoNT serotype E (BoNT/E) that is produced from C. botulinum and C. butyricum has been classified into six subtypes designated E1 to E6 (11). Subtypes E1, E2, E3, and E6 are C. botulinum, and subtypes E4 and E5 are C. butyricum strains.…”

“…Subtypes E1, E2, E3, and E6 are C. botulinum, and subtypes E4 and E5 are C. butyricum strains. The differences of amino acid sequences among the six subtypes range from 1.0z to 5.3z (11,12). C. butyricum BoNT/E is neutralized by botulinum type E antitoxin (13).…”