Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR–Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

Toxic hijack of a cell signaling pathway: The pathogen Clostridium difficile colonizes the human colon when the normal microbiota is disrupted, often after antibiotic treatment. It is a leading cause of hospital-acquired diarrhea, especially among elderly patients. Chen et al. describe a 2.5-Å-resolution crystal structure that shows how a major virulence factor in C. difficile , toxin B (TcdB), binds to the G protein–coupled receptor Frizzled (FZD). This receptor activates the Wnt signaling pathway, which regulates homeostasis of the colonic epithelium. Surprisingly, TcdB uses a lipid cofactor to recognize FZD. This cofactor replaces a lipid normally associated with the Wnt ligand that binds FZD to activate signaling. Inhibiting the Wnt pathway likely plays a role in C. difficile pathology. Science , this issue p. 664

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9–mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific “activator” for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity. Shiga and Shiga-like toxins (Stxs) are a family of bacterial toxins and key virulence factors for the bacteria Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC), which cause food poisoning throughout the world. Ricin is a plant toxin and a potential bioterrorism agent. Stxs recognize the host receptor glycolipid Gb3, while ricin utilizes a broad range of glycans as receptors. Here, we carried out genome-wide loss-of-function CRISPR-Cas9 screens using human cells to identify factors required for Stxs and ricin. Besides host factors previously known to be involved in the action of these toxins, our screens revealed three previously poorly characterized Golgi proteins: LAPTM4A, which is specifically required for Stxs, and TMEM165 and TM9SF2, which are required for both Stxs and ricin. Further characterization demonstrates that LAPTM4A is specifically required for biosynthesis of the glycolipid Gb3, potentially acting as an “activator” protein for the glycosyltransferase that synthesizes Gb3, whereas TMEM165 and TM9SF2 are likely required to maintain a proper environment within the Golgi for optimal activity of glycosyltransferases. These findings provide mechanistic insights to glycolipid biosynthesis and regulation of glycosylation levels in the Golgi and also reveal novel therapeutic targets for preventing Stxs and ricin intoxication.