Fig. 1: The Dot/Icm effector LegK3 inhibits caspase activation in eukaryotic cells.

A, B The Δ7 mutant has lost the ability to inhibit apoptosis. HeLa cells were infected with wild-type, dotA^-, or one of the L. pneumophila chromosomal deletion mutants (Δ 2, Δ3, Δ4, Δ6, and Δ7) at an MOI of 10 for 3 h. Cells were lysed and the levels of Caspase-3/-7 in the lysates were detected by immunoblotting using antibodies specific for Caspase-3 and Caspase-7 (A). Caspase-3/-7 activity in the supernatant was determined by the substrate Ac-DEVD-pNA (B). C, E. Infection with the L. pneumophila Δ7 mutant caused significantly higher percentage of apoptosis. Cells infected with the indicated L. pneumophila strains for 3 h were determined for apoptosis by measuring the ATP levels (C) or by TUNEL staining (D, E). Representative images of L. pneumophila-infected cells that were TUNEL-positive or -negative (D). For quantitation, at least 100 infected cells were counted for each sample (E). Bar, 5 μm. F–H. LegK3 suppresses host apoptosis. HeLa cells were transfected with plasmids expressing effectors coded in the deleted region of the Δ7 mutant. The activation of Caspase-3/-7 was assessed either by immunoblotting using anti-Caspase-3/-7 antibodies (F) or by measuring the cleavage of the substrate Ac-DEVD-pNA (G). Cell viability of the transfected cells was evaluated by determining the ATP levels (H). Data in A and F are representative from three independent experiments. For B, C, E, G, and H, data shown are mean ± SD from three replicates. Unpaired two-tailed Student’s t tests were performed. Source data are provided as a Source Data file.