Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20150901
Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced ...[more]
Project description:Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified as Clostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains of C. botulinum group I and C. sporogenes clearly separated these two species into discrete clades which showed ?93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143 C. sporogenes clade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bont/B2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-bearing plasmid (pCLD), while two different strains belonging to the C. botulinum clade carried bont/B2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification.
Project description:In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements.
Project description:Botulinum neurotoxins (BoNTs) can be used therapeutically to treat a wide range of neuromuscular and neurological conditions. A collection of natural BoNT variants exists which can be classified into serologically distinct serotypes (BoNT/B), and further divided into subtypes (BoNT/B1, B2, …). BoNT subtypes share a high degree of sequence identity within the same serotype yet can display large variation in toxicity. One such example is BoNT/B2, which was isolated from Clostridium botulinum strain 111 in a clinical case of botulism, and presents a 10-fold lower toxicity than BoNT/B1. In an effort to understand the molecular mechanisms behind this difference in potency, we here present the crystal structures of BoNT/B2 in complex with the ganglioside receptor GD1a, and with the human synaptotagmin I protein receptor. We show, using receptor-binding assays, that BoNT/B2 has a slightly higher affinity for GD1a than BoNT/B1, and confirm its considerably weaker affinity for its protein receptors. Although the overall receptor-binding mechanism is conserved for both receptors, structural analysis suggests the lower affinity of BoNT/B2 is the result of key substitutions, where hydrophobic interactions important for synaptotagmin-binding are replaced by polar residues. This study provides a template to drive the development of future BoNT therapeutic molecules centered on assessing the natural subtype variations in receptor-binding that appears to be one of the principal stages driving toxicity.