MLKL and infection: To ensure the role of OspD3 in infected cells, we measured the levels of cytotoxicity and MLKL phosphorylation in HT29, HeLa (cervix epithelial cells), HaCaT (skin keratinocytes), and HCT116 (colon epithelial cells) cells infected with Shigella WT, S325, or ΔospD3. Upon infection with ΔospD3, HT29, but not the other cell lines, exhibited higher cytotoxicity and MLKL phosphorylation than cells infected with WT (Fig EV2A and B).