The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements.

Theresa J Smith,Gary Xie,Patricia A Caballero,Charles H D Williamson,Fabrizio Anniballi,Karen K Hill,Bruna Auricchio,Paul Keim,Rafael A Fernández,Jason W Sahl,Shannon L Johnson

doi:10.3389/fmicb.2021.566908

Abstract

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

Highlights

Botulinum neurotoxins (BoNTs) are a worldwide public health issue and are listed as Tier 1 Select Agents due to their potential to pose a severe threat to human and animal health (Federal Select Agent Program Select Agents and Toxins List
The occurrence of human and animal botulism is directly linked to exposure to BoNTs or BoNT-producing bacteria that are resident in the environment or have been introduced via exported foods or other materials
It is known that the bont/A2 gene is a mosaic of the bont/A1 and bont/A3 genes (Hill et al, 2007), and comparisons of bont/F genes as part of this study has revealed homologous recombination (HR) events among these subtypes as well

Summary

Introduction

Botulinum neurotoxins (BoNTs) are a worldwide public health issue and are listed as Tier 1 Select Agents due to their potential to pose a severe threat to human and animal health (Federal Select Agent Program Select Agents and Toxins List). Seven toxin types (A-G) have been determined using these methods Four of these serotypes, BoNT/A, BoNT/B, BoNT/E, and BoNT/F, have been definitively linked with human botulism. BoNT/A, BoNT/B, BoNT/E, and BoNT/F, have been definitively linked with human botulism While these toxins show commonalities in protein structure and activity, genetic sequencing of the toxin serotypes has revealed that they differ by 35–70% in amino acid sequence. The subtypes are labeled with a number following the toxin type, such as BoNT/A1 or BoNT/F5 This diversity among BoNT proteins contrasts with tetanus toxin, (TeNT), which is closely related to the BoNTs in structure and mechanism of action but differs in both its singularity and a lack of non-toxic complex proteins

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Conclusion