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Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster.

Doxey AC, Lynch MD, Müller KM, Meiering EM, McConkey BJ - BMC Evol. Biol. (2008)

Bottom Line: Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity.Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada. acdoxey@uwaterloo.ca

ABSTRACT

Background: Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.

Results: The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.

Conclusion: Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.

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Genomic location of flagellin CBO0798 and regions of sequence similarity with CNTs. A) Genomic context of the neurotoxin gene cluster for C. botulinum A. str. Hall. B) Domain structure of CNTs and regions of detected similarity with NTNH (region I) and BoNT/A (region 2) according to SSEARCH. A consensus CNT secondary structure based on a multiple sequence alignment is indicated below the schematic, with black lines representing alpha helices and grey lines representing beta sheets. C) The structure of BoNT/A (PDB ID 3BTA) with region II highlighted in red. D) A Smith-Waterman alignment of region II from C. butyricum BoNT/E and CBO0798.
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Figure 2: Genomic location of flagellin CBO0798 and regions of sequence similarity with CNTs. A) Genomic context of the neurotoxin gene cluster for C. botulinum A. str. Hall. B) Domain structure of CNTs and regions of detected similarity with NTNH (region I) and BoNT/A (region 2) according to SSEARCH. A consensus CNT secondary structure based on a multiple sequence alignment is indicated below the schematic, with black lines representing alpha helices and grey lines representing beta sheets. C) The structure of BoNT/A (PDB ID 3BTA) with region II highlighted in red. D) A Smith-Waterman alignment of region II from C. butyricum BoNT/E and CBO0798.

Mentions: A comprehensive analysis of pairwise sequence similarities was performed for all proteins encoded within the C. botulinum (strain Hall A, ATCC 3502) genome [20], in an attempt to identify distant homologues of CNTs and possible sequence remnants of the evolutionary process by which CNTs originated. This initial analysis was limited to a single genome for a more sensitive detection of pairwise homologies using a restricted database, however subsequent searches were also performed using all available clostridial genomes. For the 3615 proteins encoded within C. botulinum (strain Hall A, ATCC 3502) [20], a 'heat map' of pairwise sequence similarity was constructed (see Methods) (Figure 1). For each pairwise alignment, the E-value and percentile rank relative to all other pairwise alignments was calculated using SSEARCH [21]. When compared by percentile rank, the neurotoxin gene cluster stood out as a "hot spot" of local pairwise sequence similarities. The neurotoxin gene cluster can be seen as a distinct cluster of high-scoring pairs in the centre of the heat map region in Figure 1A. Based on both the percentile ranks and E-values for the pairwise alignments corresponding to these genes (Figure 1B), there are clear sequence similarities between multiple sequences within this region, including BoNT/A, non-toxic non-hemagglutinin (NTNH), the adjacent hemagglutinin (HA) components and the adjacent CBO0798 gene encoding a flagellin protein (NCBI accession YP_001253335). BoNT/A and NTNH produced the top-scoring alignments with each other out of 3615 proteins in C. botulinum strain A (E = 1e-22, 9e-24), an expected result given previously identified sequence similarities between BoNTs and NTNH [22] as well as their virtually identical domain architecture as identified by the NCBI's conserved domain database annotation (e.g., for NCBI IDs ABP48106 and BAA90660). Surprisingly, the next highest match in both cases corresponds to the CBO0798 flagellin gene located immediately upstream of the neurotoxin gene cluster (Figure 2A). The associated E-values were 0.041 and 0.42 for BoNT/A and NTNH, respectively (Figure 1B). CBO0798 aligned with NTNH and BONT/A in two different CNT regions (I and II) (Figure 2B, Additional Files 1, 2). Additional searches using the sequences of CNTs from other strains also identified CBO0798 as the most consistent top ranked hit out of all C. botulinum strain A proteins with sequence identities between CNTs and CBO0798 ranging from 20–24%, and the strongest alignments involving region II of CNTs (Additional File 1).


Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster.

Doxey AC, Lynch MD, Müller KM, Meiering EM, McConkey BJ - BMC Evol. Biol. (2008)

Genomic location of flagellin CBO0798 and regions of sequence similarity with CNTs. A) Genomic context of the neurotoxin gene cluster for C. botulinum A. str. Hall. B) Domain structure of CNTs and regions of detected similarity with NTNH (region I) and BoNT/A (region 2) according to SSEARCH. A consensus CNT secondary structure based on a multiple sequence alignment is indicated below the schematic, with black lines representing alpha helices and grey lines representing beta sheets. C) The structure of BoNT/A (PDB ID 3BTA) with region II highlighted in red. D) A Smith-Waterman alignment of region II from C. butyricum BoNT/E and CBO0798.
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Figure 2: Genomic location of flagellin CBO0798 and regions of sequence similarity with CNTs. A) Genomic context of the neurotoxin gene cluster for C. botulinum A. str. Hall. B) Domain structure of CNTs and regions of detected similarity with NTNH (region I) and BoNT/A (region 2) according to SSEARCH. A consensus CNT secondary structure based on a multiple sequence alignment is indicated below the schematic, with black lines representing alpha helices and grey lines representing beta sheets. C) The structure of BoNT/A (PDB ID 3BTA) with region II highlighted in red. D) A Smith-Waterman alignment of region II from C. butyricum BoNT/E and CBO0798.
Mentions: A comprehensive analysis of pairwise sequence similarities was performed for all proteins encoded within the C. botulinum (strain Hall A, ATCC 3502) genome [20], in an attempt to identify distant homologues of CNTs and possible sequence remnants of the evolutionary process by which CNTs originated. This initial analysis was limited to a single genome for a more sensitive detection of pairwise homologies using a restricted database, however subsequent searches were also performed using all available clostridial genomes. For the 3615 proteins encoded within C. botulinum (strain Hall A, ATCC 3502) [20], a 'heat map' of pairwise sequence similarity was constructed (see Methods) (Figure 1). For each pairwise alignment, the E-value and percentile rank relative to all other pairwise alignments was calculated using SSEARCH [21]. When compared by percentile rank, the neurotoxin gene cluster stood out as a "hot spot" of local pairwise sequence similarities. The neurotoxin gene cluster can be seen as a distinct cluster of high-scoring pairs in the centre of the heat map region in Figure 1A. Based on both the percentile ranks and E-values for the pairwise alignments corresponding to these genes (Figure 1B), there are clear sequence similarities between multiple sequences within this region, including BoNT/A, non-toxic non-hemagglutinin (NTNH), the adjacent hemagglutinin (HA) components and the adjacent CBO0798 gene encoding a flagellin protein (NCBI accession YP_001253335). BoNT/A and NTNH produced the top-scoring alignments with each other out of 3615 proteins in C. botulinum strain A (E = 1e-22, 9e-24), an expected result given previously identified sequence similarities between BoNTs and NTNH [22] as well as their virtually identical domain architecture as identified by the NCBI's conserved domain database annotation (e.g., for NCBI IDs ABP48106 and BAA90660). Surprisingly, the next highest match in both cases corresponds to the CBO0798 flagellin gene located immediately upstream of the neurotoxin gene cluster (Figure 2A). The associated E-values were 0.041 and 0.42 for BoNT/A and NTNH, respectively (Figure 1B). CBO0798 aligned with NTNH and BONT/A in two different CNT regions (I and II) (Figure 2B, Additional Files 1, 2). Additional searches using the sequences of CNTs from other strains also identified CBO0798 as the most consistent top ranked hit out of all C. botulinum strain A proteins with sequence identities between CNTs and CBO0798 ranging from 20–24%, and the strongest alignments involving region II of CNTs (Additional File 1).

Bottom Line: Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity.Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada. acdoxey@uwaterloo.ca

ABSTRACT

Background: Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.

Results: The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.

Conclusion: Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.

Show MeSH
Related in: MedlinePlus