The L. pneumophila clpP mutant was impaired in both cytotoxicity against amoebae A. castellanii and growth on amoebae plates. (A) Growth of L. pneumophila LpΔclpP mutant in the amoebae plate test was impaired. L. pneumophila wild-type strain JR32, LpΔclpP mutant, clpP complemented strain or dotA mutant were spotted respectively in tenfold serial dilutions onto BCYE agar plates containing A. castellanii. The plates were incubated at 37°C for 5 days. (B) Cytotoxicity of L. pneumophila against amoebae A. castellanii was quantified by flow cytometry and (C) detected by PI staining 24 h post infection. The infection was performed using the wild-type strain JR32, LpΔclpP mutant, clpP complemented strain or dotA mutant at an MOI of 100. For fluorescence microscopy, amoebae cells in each well of 24-well plate were stained. The data shown are representative of at least two independent experiments.