Fig 2: Host cells depleted of executioner caspases are less resistant to intracellular bacterial growth.Parental HeLa or commercial caspase-3 KO (C3) cells (Abcam) were nucleofected with Cas9/guide RNA ribonucleoproteins (RNPs) targeting caspase-7 before enriching for deletion lines by limited dilutions. Knockout was confirmed by immunoblot using anti caspase-3, caspase-7 and α-tubulin antibodies (A). B caspase-deficient or parental HeLa lines were treated with staurosporine (STS) for 7 h before lysis and measuring DEVDase activity using DEVD-pNA. Indicated HeLa lines were infected with WT Listeria at MOI 0.1 for 16 h +/− TNF-α before the cells were lysed and DEVDase activity was measured (C), and CFU were calculated from bacterial growth curves (D and E), as shown in Figure S1. Averages +/− SEM of three independent experiments are shown. THP-1 cells were nucleofected with Cas9/guide RNPs targeting caspase-3 or -7 before cloning by limited dilutions and assessment by western blot (F). Cells of indicated genotype were infected with Lm at the MOI of 0.1 for 16 h +/− TNF-α, then the cells were lysed and DEVDase activity was assessed using fluorescent DEVD-fmk (G), and CFU were determined as above (H). WT and caspase-7 KO THP-1 cells were additionally infected with SL1344 at a MOI of 0.001 + /- TNF-α for 16 h before intracellular CFU were calculated from growth curves as shown in Figure S1G (I). Averages +/− SEM of three independent experiments are shown and significant differences between groups are indicated by asterisks. P-values are * < 0.05, ** < 0.01 and *** < 0.005.

Fig 3: A Lm lines secreting caspases-3 and -7-uncleavable LLO or caspase-3-uncleavable Iap are more virulent in HeLa cells.Culture supernatants of ΔLLO Lm, transfected with indicated pIMK2-LLO constructs (A), were treated with caspase-3 or caspase-7 for 4 h (B). Secretion and caspase-mediated cleavage of LLO were assessed by immunoblots. Detection of Iap served as loading control in (A). C Human red blood cells were treated with serial dilutions (C) or caspase-treated (D) Lm supernatants containing LLOwt or LLOc3/7 for 15 min at 37 °C. Hemoglobin release was measured spectrophotometrically at 405 nm wavelength and average +/− SEM of three independent experiments is presented. E HeLa cells were infected with indicated Lm mutants +/− zVAD +/− TNF-α treatment before the intracellular bacterial load was assessed by growth curves assay. Arrows indicate the differences in delay times to the zVAD controls to reach the threshold OD of 0.1 that is plotted in (F). Average +/− SEM of the delay time normalized to zVAD controls in three independent experiments is shown. G Lm culture supernatants of lines secreting caspase-3-uncleavable (IAPc3) or wild-type Iap (IAPwt) were treated with caspase-3 for 4 h and cleavage of Iap was assessed by immunoblot. H HeLa cells were infected with indicated Lm mutants +/− TNF-α before the intracellular CFU were calculated from growth curves. Average +/− SEM of three independent experiments is shown. Significant differences between indicated groups are marked by asterisks. P-values are * < 0.05, **< 0.01 and ***< 0.005.

Fig 4: Executioner caspases activity is induced by Listeria monocytogenes infection in viable host cells that inhibits intracellular growth.A HeLa cells were treated with staurosporine (STS, 0.1 μg/ml) or infected with virulent Lm (WT, strain 10403S), listeriolysin O deficient Lm (ΔLLO) or Salmonella enterica serovar Typhimurium (SL1344) at a MOI of 1 for 1 h before extracellular bacteria were removed by gentamicin treatment. After further culture, the cells were lysed at indicated time points before caspase activity in the lysates was assessed using the chromogenic dye DEVD-pNA. Averages +/− SEM of three independent experiments are shown. HeLa cells infected with indicated bacteria as above +/− DEVD (20 μM), zVAD (20 μM) or STS (0.1 μg/ml) treatment were lysed at the indicated time points and their lysates were assessed by western blot for cleaved caspases-9 and -7 (Cl. casp9 and casp7), as well as the caspase-3-cleavage fragment of Parp1 (cl. parp1, B) or cleaved caspase-3 (cl. casp3) after 16 h (C). α−tubulin served as loading control. HeLa cells were infected with Lm WT as above or left non-infected (non-inf) for 16 h +/− TNF-α (10 ng/ml) before measuring caspase-9, caspase-3 and caspase-7 cleavage (D), as well as activation of caspase 8 (E) by immunoblot. In all panels, representative blots of three independent experiments are shown. DEVDase activity (F), MTS metabolic activity (G) and LDH release (H) were assessed in HeLa cells infected as above for 16 h with Lm WT, Lm ΔLLO or in uninfected cells +/− TNF-α (10 ng/ml) +/− zVAD (20 μM) treatment. Average +/− SEM of three independent experiments is shown. I HeLa cells were infected with Lm WT as above +/− DEVD-fmk (20 μM), zVAD (20 μM) or TNF-α (10 ng/ml) treatment. At indicated times, cells were lysed, and Lm were enumerated by CFU assay. Average +/− SEM of three independent experiments is presented. Asterisks indicate significant differences to untreated controls. P-values are * < 0.05, ** < 0.01 and *** < 0.005.