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

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.

First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy, Species Genetic Diversity, and Virulence Related to Gas Gangrene

“…Prophage 1 and 3 matched to phiCD38-2/phiCD111/phiCD146 and phiCD6356 phages respectively, reported earlier from C. difficle ( Fortier and Moineau, 2007 ; Sekulovic et al, 2014 ). On the other hand, prophages (Geobac_E2/Bacill_phIS3501/Coryne_StAB) identical to prophage 4 are also reported in the genus Clostridium ( Smith et al, 2021 ) as well as in other species such as Shigella ( Muthuirulandi Sethuvel et al, 2019 ) and Corynebacterium (NC_048780). Phage vB_CpeS-CP51 (prophage 2), has been initially recognized as a temperate bacteriophage of C. perfringens ( Gervasi et al, 2013 ) and was later reported also in C. chauvoei ( Frey and Falquet, 2015 ).…”

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense

“…All gene clusters contain a gene that encodes a non-toxin non-hemagglutinin protein, which is believed to play a role in protecting the toxin molecule as it traverses the digestive system of the host. The two types of gene clusters differ in that one type contains three hemagglutinin genes and the other contains three orfX genes of unknown function [ 3 ].…”

“…Comparisons of identical bont gene clusters within plasmids versus those that have integrated onto the chromosome affords us the opportunity to better understand the extent and composition of transferred material and identify surrounding genes or genetic elements that may be involved in the specificity of such transfers. With a few exceptions, the transferred material is limited to DNA sequences of ~17–33 kb containing the bont gene cluster and a few surrounding genes [ 3 ].…”

Infant Botulism: Checklist for Timely Clinical Diagnosis and New Possible Risk Factors Originated from a Case Report and Literature Review

“…BoNTs are produced by anaerobic spore-forming bacteria belonging to the genus Clostridium and called BoNT-producing clostridia. Clostridium botulinum, and rarely Clostridium baratii and Clostridium butyricum, have been recognized as responsible for producing Toxins 2021, 13, 860 2 of 11 toxins involved in human botulism [3]. Recently, whole genome sequencing and molecular investigations revealed that some strains are capable of producing BoNT/B clustered as Clostridium sporogenes, which is notably resembled as a non-toxigenic strain related to proteolytic C. botulinum [3].…”

“…Clostridium botulinum, and rarely Clostridium baratii and Clostridium butyricum, have been recognized as responsible for producing Toxins 2021, 13, 860 2 of 11 toxins involved in human botulism [3]. Recently, whole genome sequencing and molecular investigations revealed that some strains are capable of producing BoNT/B clustered as Clostridium sporogenes, which is notably resembled as a non-toxigenic strain related to proteolytic C. botulinum [3]. Generally, strains produce one type of BoNT; however, some can produce two or three toxins (or mosaic toxins) or produce one toxin and harbor a silent gene [4].…”