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The 25 kDa H_CN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

ABSTRACT: The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain H_N, and the C-terminal receptor binding domain H_CC are largely resolved, but that of the H_CN domain sandwiched between H_N and H_CC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their H_CN domains or swapping H_CN domains between each other. Both deletion and replacement of TeNT H_CN domain by H_CNA and H_CNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping H_CN domains between BoNT/A and B hardly impaired their biological activity, but substitution with H_CNT did. Binding assays revealed that in the absence of H_CN, not all receptor binding sites are equally well accessible. In conclusion, the presence of H_CN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the H_CN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that H_CNT plays a different role in the intoxication mechanism of TeNT.

SUBMITTER: Deppe J

PROVIDER: S-EPMC7760224 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

Deppe Julian J Weisemann Jasmin J Mahrhold Stefan S Rummel Andreas A

Toxins 20201126 12

The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain HN, and the C-terminal receptor binding domain HCC are largely resolved, but that of the HCN domain sandwiched between HN and HCC has remained uncl ...[more]

PMID: 33255952

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Project description:BackgroundClostridial 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.ResultsThe 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.ConclusionMultiple 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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The 25 kDa H_CN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

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The 25 kDa H<sub>CN</sub> Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

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