FIG. 3.

Differential requirement for NAIP5 in sensing Legionella and Salmonella is dictated by the bacterium and not the flagellin itself. (A) Cell death was assessed by the measurement of lactate dehydrogenase (LDH) release from wild-type (B6), isogenic Naip5^−/−, or isogenic Naip5^−/− Nlrc4^−/− doubly deficient macrophages infected with either wild-type (WT) Legionella (LP02), isogenic flagellin-deficient Legionella (ΔflaA), wild-type Salmonella (LT2), or isogenic flagellin-deficient Salmonella (designated LT2 fliC/fljB⁻). Gentamicin (100 μg/ml) was added 30 min after infection to kill extracellular bacteria. (B) Cell death was assessed by LDH release from B6, Naip5^−/−, or Nlrc4^−/− macrophages infected with WT Legionella (LP02), Legionella ΔflaA, or Legionella expressing Salmonella fliC from the Legionella chromosome in place of its native flaA gene (ΔflaA::fliC). Gentamicin (100 μg/ml) was added 30 min after infection to kill extracellular bacteria. (C) IL-1β release assayed from macrophages infected with the indicated strains, as shown in panel B. Macrophages were pretreated with tripalmitoyl cysteinyl seryl tetralysine lipopeptide (Pam3CSK₄; 0.5 μg/ml) for 3.5 h to induce the expression of pro-IL-1β prior to infection. (D) Growth of ΔflaA or ΔflaA::fliC Legionella in wild-type B6, isogenic Naip5^−/−, or isogenic Nlrc4^−/− macrophages was assayed by determining the CFU at the time points indicated. *, P < 0.00. Two-way ANOVA and Bonferroni's test were used to determine the statistical significance (P) of differences in IL-1β and LDH release (B and C) against infection with ΔflaA Legionella. For each panel, data shown are from a single representative experiment of at least two that produced similar results.