Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins

doi:10.1016/j.toxicon.2015.09.011

Toxicon

Volume 107, Part A, 1 December 2015, Pages 2-8

https://doi.org/10.1016/j.toxicon.2015.09.011 Get rights and content

Highlights

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Genetic diversity of botulinum neurotoxin-producing clostridia is discussed.
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Toxin subtypes are compared and delineated.
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Mechanisms for the movement of toxin genes are explained.
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Toxin cluster types are illustrated.
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Toxin locations within genomes are highlighted.

Abstract

The recent availability of multiple Clostridium botulinum genomic sequences has initiated a new genomics era that strengthens our understanding of the bacterial species that produce botulinum neurotoxins (BoNTs). Analysis of the genomes has reinforced the historical Group I–VI designations and provided evidence that the bont genes can be located within the chromosome, phage or plasmids. The sequences provide the opportunity to examine closely the variation among the toxin genes, the composition and organization of the toxin complex, the regions flanking the toxin complex and the location of the toxin within different bacterial strains. These comparisons provide evidence of horizontal gene transfer and site-specific insertion and recombination events that have contributed to the variation observed among the neurotoxins. Here, examples that have contributed to the variation observed in serotypes A-H strains are presented to illustrate the mechanisms that have contributed to their variation.

Introduction

Clostridium botulinum is a species that is defined by the production of any one of eight serologically distinct botulinum neurotoxins designated BoNT/A-H (Peck, 2009). The toxin is a zinc metalloprotease that consists of a light chain, heavy chain and translocation domain. The three domains interact with accessory proteins of the toxin complex to assist the holotoxin in entering the bloodstream, then neuronal cells (Fujinaga et al., 2013). Once inside the neuronal cells the light chain domain cleaves specific sites within SNARE proteins. The cleavage of these proteins prevents the release of acetylcholine into the neuronal muscular junction and results in a flaccid paralysis known as botulism (Schiavo et al., 1995).

The BoNT-producing bacteria are generally called C. botulinum, but they could be considered six different species by 16S rrn comparisons (Hill et al., 2009). Group I–IV designations are used to distinguish the different species within C. botulinum. The C. botulinum Group I strains produce BoNT/A, /B, /F or /H; C. botulinum Group II strains produce BoNT/B, /E or /F; C. botulinum Group III strains produce BoNT/C or /D; C. botulinum Group IV (also designated Clostridium argentinense) strains produce BoNT/G. Group V retained the species name of Clostridium baratii for strains that produce BoNT/F and Group IV are Clostridium butyricum strains that produce BoNT/E (Popoff and Marvaud, 1999). The presence of the same toxin type in different Groups/species indicates that the toxin (and its associated genes within the toxin complex) move into different bacterial species by horizontal gene transfer (HGT).

The eight BoNTs (A-H) proteins are serologically distinct and differ by 37–70% in amino acid sequence (Dover et al., 2014b, Hill and Smith, 2013). BoNT/A, /B, /E and /F are the toxins that most commonly cause human botulism. Strains can produce a single toxin or multiple toxins, such as Ba, Bf, Bh, Ab, Af, where the capital letter designates the toxin produced in greater amounts. Six of the eight toxin types (BoNT/A, /B, /C, /D, /E and /F) have variants called subtypes. There are currently 40 recognized toxin subtypes (Fig. 1); amino acid differences may be minor (1–7% difference among type B) or major (3–36% among type F) (Hill and Smith, 2013). The subtypes are given the toxin letter designation followed by a number. For example, there are eight different subtypes of BoNT/A, designated BoNT/A1–A8. Understanding the variation within a toxin type is important to ensure the effectiveness of detection methods or the effectiveness of toxin treatments.

The BoNT protein naturally associates with several nontoxic proteins to form a toxin complex. Within the genome, the genes for these proteins form one of two different toxin gene clusters: 1) the toxin gene cluster containing a nontoxin-nonhemagglutinin (ntnh) gene, a regulatory gene (botR) (Connan et al., 2013), a gene of unknown function (p47) (Chen et al., 2008) and three predicted open reading frames – this is known as the orfX+ gene cluster; and 2) the hemagglutinin (ha+) toxin gene cluster, where three hemagglutinin genes replace the orfX1–X3 genes (Fig. 2) (Hill and Smith, 2013). Each of the toxin gene cluster types is associated with specific BoNTs. The orfX+ gene cluster is associated with four toxin genes: bont/A, /E, /F or /H and the ha+ gene cluster is associated with five toxin genes: bont/A, /B, /C, /D or /G. Only the bont/A is found within either the orfX+ or ha+ gene cluster. All other bont genes are located within one or the other of the toxin gene clusters.

The toxin type and subtype, the presence of its gene within an ha+ or orfX+ gene cluster, and its location within various bacterial species/Groups provide valuable information related to characterization of the toxin and the bacteria that produce these toxins. The same toxin gene's presence in different bacterial species illustrates insertion or HGT events. The changes in toxin sequences through recombination events can result in the creation of new subtypes. In addition, the genomic location of the toxin genes does not appear to be random and examination of the mechanisms of toxin gene insertions can assist in understanding relationships of toxin-containing strains.

HGT of the toxin gene can be facilitated by different mechanisms, including the presence of the toxin within mobile elements such as plasmids or phage, the association of the toxin with transposases or Insertion Sequence (IS) elements, or the presence of direct repeated sequences in the regions flanking the toxin gene cluster which may provide a targeting and insertion site. The mobility of the toxin gene cluster and its presence in different bacterial species provides the opportunity for the toxins to evolve within different bacterial backgrounds and environments through genetic recombination, resulting in the diverse array of toxins that have been identified. Below are descriptions of significant insertion and recombination events that have resulted in toxin diversity or the placement of toxin genes within different bacteria Groups/species. The examples presented here are not comprehensive descriptions of each toxin but rather provide examples illustrating various toxin mobility, insertion, and recombination mechanisms in multiple species of bacteria that result in botulinum neurotoxin diversity.

Section snippets

BoNT/A

BoNT/A is produced by C. botulinum Group I bacteria and currently consists of 8 subtypes (BoNT/A1–A8) that have protein differences that are greater than 15% (Kull et al., 2015, Smith, 2014). The location of the bont/A gene within a strain can be within one of two sites within the chromosome (arsC operon or oppB/brnQ operon), or at one site within large Group I plasmids (Hill et al., 2009). Comparisons of the genomic sequences in multiple strains have shown that the sites of the bont genes

Conclusion

Examination of the sequences from bont genes, toxin complex genes, and genomes from multiple C. botulinum strains has enabled us to increase our understanding of the mechanisms underlying toxin diversity. These mechanisms include the movement of genetic material using mobile genetic elements, such as plasmids and phage, and insertion and recombination events facilitated by a variety of insertion mechanisms, including simple recombination events, targeting of specific gene homologs followed by

Acknowledgments

This research was partially funded by a grant from NIH/NIAID U01 AI056493. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.

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View full text

Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins

Highlights

Abstract

Introduction

Section snippets

BoNT/A

Conclusion

Acknowledgments

Toxicon Off. J. Int. Soc. Toxinol.

J. Mol. Epidemiol. Evol. Genet. Infect. Dis.

Vaccine

Three classes of plasmid (47–63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum

Genome Biol. Evol.

Complete genome sequence of the proteolytic Clostridium botulinum type A5 (B3′) strain H04402 065

J. Bacteriol.

The type F6 neurotoxin gene cluster locus of group II Clostridium botulinum has evolved by successive disruption of two different ancestral precursors

Genome Biol. Evol.

Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature

Appl. Environ. Microbiol.

Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a sigma factor that recognizes the botulinum toxin gene cluster promoters

PloS One

Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5

PloS One

Molecular characterization of a novel botulinum neurotoxin type h gene

J. Infect. Dis.

Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component

Int. J. Syst. Bacteriol.

Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains

PloS One

Uptake of botulinum neurotoxin in the intestine

Curr. Top. Microbiol. Immunol.

GenBank Posting of Clostridium Botulinum A2B3 87 Plasmid P1_72A2B3 (AUZB01000012)

Draft genome sequence of bivalent Clostridium botulinum strain IBCA10-7060, encoding botulinum neurotoxin B and a new FA mosaic type

Genome Announc.