Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription-and replication-competent virus-like particles, with an IC 50 as low as 330 nM. Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additionalglycopeptidesaspotentialinhibitorsofcathepsinL-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection.

SummaryClostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we carried out CRISPR/Cas9-mediated genome-wide screens and identified the members of the Wnt receptor Frizzled (FZDs) family as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity toward FZD1, 2, and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signaling. FZD1/2/7 triple-knockout (KO) cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7 KO mice, combined with knock-down of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7 KO 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.

infection is the most common cause of antibiotic-associated diarrhea in developed countries. The major virulence factor, toxin B (TcdB), targets colonic epithelia by binding to the frizzled (FZD) family of Wnt receptors, but how TcdB recognizes FZDs is unclear. Here, we present the crystal structure of a TcdB fragment in complex with the cysteine-rich domain of human FZD2 at 2.5-angstrom resolution, which reveals an endogenous FZD-bound fatty acid acting as a co-receptor for TcdB binding. This lipid occupies the binding site for Wnt-adducted palmitoleic acid in FZDs. TcdB binding locks the lipid in place, preventing Wnt from engaging FZDs and signaling. Our findings establish a central role of fatty acids in FZD-mediated TcdB pathogenesis and suggest strategies to modulate Wnt signaling.