Fig. 3.
Characterization of mutants in tissue culture and mouse models of infection. (A and B) LDH release assays in BMMs. PactA-holin-lysin is a strain that expresses holin and lysin genes under the control of the actA promoter, causing Aim2-mediated pyroptosis (25). Cells were washed 30 min postinfection with media containing gentamicin for 10 min, washed three times, and incubated with gent-free media to prevent bacterial death. ND = not detected. (C) Plaque assay to measure cell-to-cell spread of AU mutants in L2 cells. (D) Plaque assay in GC mutants. UTR mutants formed plaques of decreased opacity, indicating less cytotoxicity despite cell-to-cell spread. (E) Growth curve in B6 BMMs. (F) Virulence of the mutant bacteria in CD-1 mice (i.v. dose = 105) for 48 h. Error bars represent SDs from three independent replicates. Significance was determined by unpaired t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, NS = not significant (P > 0.05). ND = not detected.