Recently, Liang Tao and his colleagues at Westlake University identified TFPI as a physiologically relevant receptor forClostridioides difficiletoxin B (TcdB) from the hypervirulent clade 2 lineages. The work, published on March 17 inCell (https://authors.elsevier.com/a/1elpeL7PXingP), has filled a major knowledge gap inC. difficileinfection pathogenesis.

C. difficileinfections are a major cause of hospital- and community-acquired diarrhea and pseudomembranous colitis globally.C. difficilecan be classified into five major evolutionary clades. Previously, Tao et al. also defined the colonic epithelial receptor for TcdB1 from the classic clade 1C. difficile. However,C. difficilestrains from clade 2 (also known as the hypervirulent clade) exclusively express two TcdB variants (TcdB2 and TcdB4) that do not recognize the classic TcdB receptor FZD. How these TcdB variants target the intestinal epithelium to initiate the damage remains unknown.

In this study, Tao and colleagues first observed that HeLa cells with FZD knocked-out were highly potent to TcdB4, suggesting the existence of an uncharacterized novel receptor. Therefore, the researchers performed a genome-wide CRISPR/Cas9 mediated knockout screen and identified tissue factor pathway inhibitor (TFPI) as a cellular receptor for TcdB4. TFPI has two dominant spliced isoforms, TFPIα and TFPIβ, and both can mediate the cellular entry of TcdB4. Moreover, TcdB specifically binds the second Kunitz domain of TFPI.

Tao next collaborated with Yanyan Li from Yigong Shi's team at Westlake. Using single-particle cryo-electron microscopy, they resolved the 3D structure of TcdB4 in complex with TFPI. Interestingly, the cryo-EM structure of TcdB4-TFPI revealed an interacting pattern similar to TcdB1-FZD2 and TcsL-SEMA6A. The finding solidifies the notion that LCTs have evolved to bind different receptors via a similar interacting region. By structural and phylogenic analysis, TcdB variants can be divided into two classes: one recognizes FZD (TcdB1/3/5), and the other recognizes TFPI (TcdB2/4).

Lastly, the researchers turned to mouse models to investigate the role of TFPI in TcdB-induced pathologyin vivo. Because complete disruption of TFPI in mice is intrauterine lethal, they generated the TFPIβ KO mice. When given an intraperitoneal injection with TcdB4 (1 μg/kg), all tested wildtype (Tfpiβ+/+) mice died rapidly within 12 hours. Strikingly, over 60% of the homozygous KO (Tfpiβ‒/‒) mice survived. Histopathological study showed that kidneys are vulnerable organs during systemic exposure to the toxin. In the colon, whichC. difficilemainly colonizes, TFPI is highly expressed in the cryptic area. The researchers built TFPI-mimicking decoys to neutralize TcdB2/4. As expected, these decoys effectively protected either cells or mice from TcdB2/4. "These results not only demonstrate TFPI as a physiologically relevant receptor for clade 2C. difficileTcdB but also provide potential therapeutic strategies againstC. difficileinfection," Tao said.

Jianhua Luo, Qi Yang, Xiaofeng Zhang, Yuanyuan Zhang, and Li Wan are co-first authors of the paper; Yanyan Li and Liang Tao (Lead contact) are corresponding authors.

This study was partially supported by the National Natural Science Foundation of China (Grant No. 31970129 and Grant No. 31800128 to L.T., Grant No. 32171205 to Y.L.), Zhejiang Provincial Natural Science Foundation of China (Grant No. LR20C010001 to L.T. and Grant No. LR20C050001 to Y.L.), Westlake Education Foundation, and Westlake Laboratory of Life Sciences and Biomedicine.