Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species

Abstract

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.

Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster

“…While the reference mapping settings utilized in our study allowed identification of protospacers with up to two mismatches and local alignment allowed some flexibility at the ends of the spacer-protospacer alignment, this approach is conservative compared with an 80% identity blastN threshold (Negahdaripour et al, 2017) and may have excluded more distant matches in other genera. Interestingly, nearly all matched plasmids were Gram-positive bacteria, and hits to plasmids in genera including Enterococcus and Paenibacillus are consistent with rare, but documented horizontal gene flows of bont gene cluster constituents (Zhang et al, 2018;Nowakowska et al, 2019). CRISPR-Cas system spacers will generally target MGEs that are most often encountered by the host strains (Shmakov et al, 2017), indicating that the plasmids present in the G1 C. botulinum and C. sporogenes mobilomes have been present long enough for CRISPR-CAS system mediated immunity to develop in certain strains.…”

“…Analysis of viral protospacers was limited to prophage present within closed G1 C. botulinum and C. sporogenes genomes, limiting insight into the broader host range targeted bacteriophage. However, matched RefSeq plasmids from other soil-dwelling Gram-positive genera included Paenibacillus and Enterococcus , which are known to possess genes homologous to bont gene cluster genes ( Zhang et al, 2018 ; Nowakowska et al, 2019 ). With further development, these data might enable enhanced risk assessment through the quantification of the normal range of horizontal gene transfer between G1 C. botulinum and other species.…”

Crystal structures of OrfX1, OrfX2 and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

“…Another intriguing finding showed that genes putatively encoding OrfX1–3 and P47 are found in genomes of a wide range of non‐BoNT‐producing bacterial species, such as Alphaproteobacteria , Bacilli , Betaproteobacteria , Cytophagia , and Gammaproteobacteria [44]. These orfX‐p47 ‐containing gene clusters show large diversity in their gene arrangement and gene content, and some are neighbouring genes encoding oral insecticidal toxins, such as delta‐endotoxins (Cry toxin), binary toxins (VIP toxin), or ABC toxins.…”

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment

“…Type B neurotoxin genes are located on a small multicopy plasmid (47-63 kb) in a ha cluster that comprises genes encoding the neurotoxin, non-toxic-non-haemagglutinin (NTNH), three haemagglutinins, and a positive regulator (botR). Type E and F neurotoxin genes are generally located on the chromosome (but for a small fraction of strains, the type E neurotoxin gene is present on a single-copy plasmid) in an orfX cluster that comprises genes encoding the neurotoxin, NTNH, and four open reading frames of unknown function [3,16,17,[23][24][25][26][27][28][29][30][31].…”