Bioinformatic discovery of a toxin family in Chryseobacterium piperi with sequence similarity to botulinum neurotoxins

Abstract

Clostridial neurotoxins (CNTs), which include botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT), are the most potent toxins known to science and are the causative agents of botulism and tetanus, respectively. The evolutionary origins of CNTs and their relationships to other proteins remains an intriguing question. Here we present a large-scale bioinformatic screen for putative toxin genes in all currently available genomes. We detect a total of 311 protein sequences displaying at least partial homology to BoNTs, including 161 predicted toxin sequences that have never been characterized. We focus on a novel toxin family from Chryseobacterium piperi with homology to BoNTs. We resequenced the genome of C. piperi to confirm and further analyze the genomic context of these toxins, and also examined their potential toxicity by expression of the protease domain of one C. piperi toxin in human cells. Our analysis suggests that these C. piperi sequences encode a novel family of metalloprotease toxins that are distantly related to BoNTs with similar domain architecture. These toxins target a yet unknown class of substrates, potentially reflecting divergence in substrate specificity between the metalloprotease domains of these toxins and the related metalloprotease domain of clostridial neurotoxins.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

“…These data demonstrated that ciA-B5 blocks the channel formation of H N A, which is a crucial step during BoNT/A intoxication. Interestingly, aa sequence analysis shows that the ciA-B5 epitope on BoNT/A1 (residues 600-616) partially overlaps with a putative channel-forming amphipathic region (Lam et al, 2018;Mansfield et al, 2019) (residues 593-686; Figures 3A, S2G, S2J, and S4A). Specifically, M39 and Y103 of ciA-B5 interact with L604, V607, V611, and Y612 of BoNT/A1, and R60 of ciA-B5 forms a salt bridge with D616 of BoNT/A1.…”

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs

“…TeNT and BoNT are also commonly indicated as clostridial neurotoxins, because until few years ago, they were known to be produced only by bacteria of the genus Clostridium . This indication is no longer correct as toxins with very similar structure and enzymatic target were recently discovered using bioinformatic methods in non‐clostridial bacterial species (Brunt, Carter, Stringer, & Peck, ; Doxey, Mansfield, & Montecucco, ; Mansfield, Adams, & Doxey, ; Mansfield & Doxey, ; Mansfield et al, ; Zhang et al, ; Zornetta et al, ). However, these “bioinformatics” toxins have not been yet associated to diseases nor it is known if they are produced at all in the natural environment (Doxey et al, ).…”

“…When expressed in Escherichia coli , the corresponding proteins were not recognised by available standard anti‐BoNT antisera. They were identified in Weissella oryzae (Mansfield et al, ; Zornetta et al, ), in strain 111 of C. botulinum (Sicai Zhang et al, ), in the genome of a strain IDI0629 of Enterococcus faecium (Brunt et al, ; S. Zhang et al, ), and in Cryseobacterium piperi (Mansfield et al, ). Most notably, both the Weissella and the Cryseobacterium sequences lack the SS bond connecting the L and HN domain.…”

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…BoNTs are produced primarily by the Gram-positive, anaerobic, spore-forming bacterial species Clostridium botulinum [31,[33][34][35]. In addition, genetically related homologs of BoNTs have also been identified in more distantly related genera, including Enterococcus, Weissella, and Chryseobacterium [17,[36][37][38][39][40][41][42]. However, while the catalytic activity of these BoNT-like LCs on SNARE proteins has been shown, there are currently no indications that these homologs act as vertebrate neurotoxins, and further research will be required to determine potential toxicity, target hosts, and biologic function.…”

“…Interestingly, recent research of novel BoNTs has found that BoNT/F5 and BoNT/FA are an exception within the F subtype LCs in that they cleave VAMP1/2 (vesicle-associated membrane protein) at unique sites, which are distinct from the cleavage sites of other type F LCs [72,73]. Interestingly, the novel putative BoNT/X, as well as BoNT homologs from organisms other than clostridia such as BoNT/Wo and BoNT/En, have been found to have unique neuronal and in some cases non-neuronal SNARE cleavage targets and sites [17,32,[36][37][38][39][40][41][42]. It is currently unknown whether novel or as yet uncharacterized BoNTs may have unique SNARE cleavage targets, which is an important consideration when using a BoNT detection method that relies on detection of specific SNARE cleavage.…”