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

Abstract

Clostridioides difficile 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 C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB 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 C. difficile. 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 C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile 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 it 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 C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

“…However, the need for bezlotoxumab to simultaneously occupy two epitopes in TcdB in order to be effective also increases its susceptibility to residue changes in TcdB variants. Epitope-1 and -2 in TcdB each consists of about 20 amino acids, and variations have been observed in many TcdB variants especially in epitope-1 16 , 20 (Supplementary Fig. 10d, e ).…”

“…These amino acid substitutions in the bezlotoxumab-binding epitopes are believed to decrease the binding affinities and neutralization potencies of bezlotoxumab 20 . For example, the neutralization efficacy of bezlotoxumab on TcdB2 is ~200-fold lower than TcdB1 16 , 19 . Consistently, we found that bezlotoxumab showed a much lower potency in blocking TcdB2 on HeLa cells compared with TcdB1 in the cell-rounding assay, and its Fab failed to show any protection at the highest concentration tested (2 µM) (Fig.…”

“…A major event in CDI epidemiology is the emergence of rapidly spreading hypervirulent strains globally in recent years, which together with a growing number of other strains produce diverse TcdB isoforms with sequence variations up to 11% 15 , 16 , 39 . Here, using complementary structural and functional studies, we identify the CSPG4-binding site in TcdB and demonstrate that CSPG4 is a common receptor for both TcdB1 and TcdB2, two largest families of TcdB isoforms responsible for over 70% of clinical isolates 16 . Furthermore, CSPG4 alone acts as a key protein receptor for the clinically important TcdB2 lacking the ability to bind FZDs.…”

“…Beyond its complex 3D structure, TcdB has greatly diversified throughout its entire primary sequence up to 11% during evolution 14 – 16 . For example, many hypervirulent fluoroquinolone-resistant lineages such as BI/NAP1/027 strains, which emerged in North America with major outbreaks in early 2000s, express a variant of TcdB (designated TcdB2) that is ~8% sequence variation from the endemic TcdB (designated TcdB1) 15 – 18 .…”

“…TcdB1 could bind FZDs and CSPG4 simultaneously, indicating that FZDs and CSPG4 are recognized by distinct regions of TcdB 21 , 27 . However, many clinically important TcdB variants, represented by TcdB2, bind CSPG4 but not FZDs, because they have residue substitutions in the FZD-binding site that abolish their binding to FZDs 14 – 16 , 27 – 29 . Moreover, the therapeutic antibody bezlotoxumab reduces binding of TcdB1 to CSPG4 in vitro 30 , suggesting CSPG4 may contribute to TcdB pathogenesis in humans.…”