Phylogenomics of 8,839Clostridioides difficilegenomes reveals recombination-driven evolution and diversification of toxin A and B

Abstract

<jats:title>Abstract</jats:title><jats:p><jats:italic>Clostridioides difficile</jats:italic> is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB) is essential for <jats:italic>C. difficile</jats:italic> pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis 8,839 <jats:italic>C. difficile</jats:italic> strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 <jats:italic>tcdA</jats:italic> and 8,022 <jats:italic>tcdB</jats:italic> genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of <jats:italic>C. difficile</jats:italic>. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in <jats:italic>C. difficile</jats:italic> evolution. The application of toxin subtyping is then validated by classifying 351 <jats:italic>C. difficile</jats:italic> clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of <jats:italic>C. difficile</jats:italic> toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of <jats:italic>C. difficile</jats:italic>.</jats:p>

Strain variation inClostridioides difficiletoxin activity associated with genomic variation at both PaLoc and non-PaLoc loci

“…Additionally, the species has had multiple independent losses of the PaLoc (17), with our results indicating that even strains harboring an intact PaLoc may evolve to have decreased toxin activity. The C. difficile strains with high toxin activity may have success by shaping a hostile .…”

“…Indeed, there are prolific toxigenic and non-toxigenic strains of C. difficile. Additionally, the species has had multiple independent losses of the PaLoc (17), with our results indicating that even strains harboring an intact PaLoc may evolve to have decreased toxin activity. The C. difficile strains with high toxin activity may have success by shaping a hostile metabolic state in the host gut that these bacteria are able to uniquely exploit (18) or its more severe, inflammatory infection which results in diarrhea and therefore increased transmission.…”

“…One possibility is that toxin activity itself may not be the most critical aspect of the toxin upon which evolution is acting, with other aspects such as toxin immunogenicity potentially evoking a stronger selection pressure. Toxin that evades immune recognition could lead to longer infections and therefore increased transmission, so the strongest selective pressure may be at the surface domains of the toxin proteins rather than on regulators of toxin activity (19). For example, we observed multiple missense variants on the surface of tcdB in this isolate collection, including a glutamic acid 329 to glycine missense variant and threonine 430 to alanine.…”

Using synthetic activity to design ultra-potent antibody cocktails

“…In vitro , these cytoskeletal changes can be measured in real-time as a change in impedance using cells cultured on a surface embedded with microelectrodes 49,50 . TcdB1 is the TcdB toxin type most frequently present in worldwide C. difficile isolates from infected patients 51 . The source of TcdB1 for these analyses was ribotype (RT) 087 (type strain 10463).…”

“…96% of the TcdB genes present in that dataset were defined as belonging to groups 1, 2, 3, and 5. Group 1 is the most clinically prevalent group worldwide, and group 2 is expressed by C. difficile ribotype 027, the single most prevalent strain type in the United States, and second most prevalent in Europe 3,51,53,54 .…”