FIG 4.

Replication of PPIase mutant strains in A. castellanii. A. castellanii was infected with different L. pneumophila mutant strains. After 2 h, 24 h, and 48 h, the amoebae were lysed and dilutions were plated out on BCYE agar plates. (A) Infection with the wild-type strain (WT), the ΔdotA mutant (ΔdotA), the mip-negative strain (Δmip), the ppiB-negative strain (ΔppiB), and the mip- and ppiB-negative double mutant strain (ΔΔ). The PPIase mutants showed decreased intracellular growth. (B) Complementing the single-knockout mutants with the respective gene restored the wild-type phenotype. Shown are the means and standard deviations from three experiments performed in triplicate. Statistical significance was calculated by an unpaired Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant).