Fig 1. N-linked Glycans are required for the cell targeting of the TcdA1^W14 pentamer.

(A) Five mammalian cell lines were treated with the indicated doses of PTC3^W14 for 24 h. Cell viability was measured using CCK-8 assays. The PTC3^W14 concentration that leads to the death of 50% of cells is defined as IC₅₀. (B) HeLa-Cas9 cells transduced with a non-targeting sgRNA were treated with 5 nM PTC3^W14 for 0, 4 and 8 h. Cells were fixed, permeabilized and stained with Alexa 568-phalloidin (red) and DAPI (blue). Representative fluorescence and bright field micrographs are shown. White arrows indicate polymerized F-actin. Black arrows indicate membrane blebs. Scale bars, 5 μm. (C) Schematic drawing of the screening approach for host factors that are critical for the intoxication of PTC3^W14. HeLa cells stably expressing Cas9 (HeLa-Cas9) were transduced with lentiviral GeCKO v.2 sgRNA libraries. These cells were exposed to increased doses of PTC3^W14 (5, 10 and 20 nM). Cells that were not treated with PTC3^W14 were served as controls. Total genomic DNA from 2×10⁷ selected and control cells was used for sequencing. The enriched sgRNAs were sequenced by NGS, followed by MAGeCK analysis. (D) Genes identified in the screens with PTC3^W14 treatment were ranked based on to the MAGeCK p values. MAN1A2, MGAT1, and MGAT2 were identified as the key genes for PTC3^W14 recognition of HeLa cells. (E) The efficiency of gene knockout was determined by Western blot analysis with the indicated antibodies. GAPDH served as the loading control. (F-H) MAN1A2-KO, MGAT1-KO, and MGAT2-KO HeLa-Cas9 cells were treated with the indicated doses of PTC3^W14 for 24 h. Cell viability was measured using CCK-8 assays. (I-J) HeLa-Cas9-sgCon cells were pretreated with the indicated doses of Kifunensine for 12 h, and then exposed to 20 nM PTC3^W14. Representative bright field micrographs were shown (I). Cell viability was measured using CCK-8 assays (J). All error bars indicate mean ± SD. Each of the experiments was repeated three times.