Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

Abstract

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens

“…Subsequently, the cells are exposed to toxins as a phenotypic selection, usually through multiple rounds, to minimize contaminants. The surviving cells at the end of the selection can be expanded and analyzed using advanced sequencing techniques to determine the specific genes involved (Figure 2) [31][32][33][34][35]. The RNAi screen is easy to handle and can be applied to multiple cell lines to achieve high genome coverage.…”

Isofagomine Inhibits Multiple TcdB Variants and Protects Mice from Clostridioides difficile-Induced Mortality

“…Furthermore, TcdB variants have been shown to use different receptors to gain entry into mammalian host cells, including the CSPG4, Frizzled, and TFPI protein receptors. 20,21 Given that multiple TcdA and TcdB variants have been identified, it is essential to evaluate TcdA and TcdB-targeted therapeutics for the inhibition of multiple variants.…”

“…Two recent studies have proposed grouping TcdB toxin variants into either 8 or 12 subtypes (TcdB1-B12). , Compared to TcdB, TcdA showed far fewer variations in both studies. , Sequence variations in the toxin genes could influence receptor binding, specificity toward Rho GTPases, antigenicity, and overall toxicity. Furthermore, TcdB variants have been shown to use different receptors to gain entry into mammalian host cells, including the CSPG4, Frizzled, and TFPI protein receptors. , Given that multiple TcdA and TcdB variants have been identified, it is essential to evaluate TcdA and TcdB-targeted therapeutics for the inhibition of multiple variants.…”

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin

“…It was shown that the CROPs domain of TcdA was capable of binding the trisaccharide Gal-α1,3-Gal-β1,4-GlcNAc and thus considered as a domain responsible for binding carbohydrate moieties on cell surface 36,37 . In recent years, multiple protein receptors for LCTs were identified, including low-density lipoprotein receptor-related protein 1 (LRP1) for TpeL 38 , low-density lipoprotein receptor (LDLR) family proteins for TcdA and Tcnα [39][40][41][42] , chondroitin sulfate proteoglycan 4 (CSPG4), poliovirus receptorlike 3 (PVRL3), Frizzled proteins (FZDs), and tissue factor pathway inhibitor (TFPI) for TcdB 33,[43][44][45][46] , Semaphorin 6 A and 6B (SEMA6A/6B) for TcsL 47,48 , and transmembrane serine protease 2 (TMPRSS2) for TcsH 49 . Many of them were structurally validated to bind LCTs independent of the CROPs 33,48,50,51 , raising the speculation that the CROPs in LCTs are not solely for host recognition but have uncharacterized functions.…”