Fig. 2: A CRISPR-Cas9 screen identifies TFPI as a receptor for TcdB4.
a Schematic diagram of the CRISPR-Cas9 screen process. b Genes identified by NGS were analyzed with the MAGeCK program61 and plotted based on the log2 value of fold change of NGS reads and statistical significance (shown as log10 value of RRA p-value and plotted as the y-axis). The genes involved in the GPI (glycosylphosphatidylinositol) biosynthetic pathway are colored red. c Schematic diagram of human TFPIβ (TFPI), a GPI-anchored protein with two BPTI/Kunitz protease inhibitor domains (K1 and K2). N, N-termini; C, C-termini. HeLa (d) or A549 (e) KO cells lacking TFPI, TFPI2 (a homolog of TFPI), PIGS, or PIGV were generated via the CRISPR-Cas9 approach. UGP2-KO cells were also analyzed as a control. Cells were exposed to recombinant TcdB4.2 for 24 h. Their CR50 values are normalized to WT and plotted in a bar-chart (f). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t-test, two-sided). g–i HeLa (g) or 5637 (h) cells overexpressing triple-HA-tagged TFPI, TFPI2, or mouse TFPI (mTFPI) via lentiviral transduction were exposed to TcdB4.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their CR50 values are normalized to WT and plotted in a bar-chart (i). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t-test, two-sided). j Binding of TcdB4.2 (500 nM) to Fc-tagged TFPI, TFPI2, and mTFPI (immobilized onto capture biosensors) was examined using biolayer interferometry (BLI) assays. Fc-tagged extracellular domains of FZD2 (CRD2), SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. k HeLa cells were exposed to either TcdB4.2 alone (4 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI at the indicated molar ratios (1:250 ~ 1:20,000) on ice for 1 h. The percentage of cell-rounding at 6 h incubation was plotted. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t-test, two-sided). l HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB2.1, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressing TcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations are shown in Supplementary Fig. 8a. Their CR50 values were normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). m–p HeLa-WT, two CSPG4 KO single clones (CSPG4#1 and CSPG4#8), and two CSPG4/TFPI double KO cells (CSPG4#1-TFPI-KO and CSPG4#8-TFPI-KO) were exposed to TcdB4.2 (m), TcdB2.1 (n), or TcdB2.2 (o) for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their relative CR50 values are plotted in a bar-chart (p). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t-test, two-sided). q HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB2.1, TcdB2.2, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressingTcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations and are shown in Supplementary Fig. 8e. Their CR50 values are normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t-test, two-sided). Source data are provided as a Source Data file.
