Figure 5. ClpA is responsible for the decreased level of DotL observed in the absence of IcmS/IcmW.

(A) Stability of DotL in the absence of IcmS and ClpA. L. pneumophila strains were grown in broth and cells were collected at various stages of growth. Whole cell extracts were analyzed by Western blotting using antibodies specific to the proteins listed beneath each panel. Optical densities (600 nm) of the cultures were similar to those described in Figure 4. Strains used were: wild-type plus vector (JV1139), ΔicmS plus vector (JV5156), ΔicmS ΔclpA::CmR plus vector (JV6296) and the ΔclpA::CmR plus vector (JV6503). Western blots for ICDH served as a loading control.

(B) Deletion of ClpA does not suppress the intracellular growth defect of the ΔicmS mutant in U937 cells. Strains used were wild-type plus vector (JV1139, filled squares), ΔclpA::CmR plus vector (JV6503, open inverted triangles), ΔicmS plus vector (JV5156, open circles), Lp03 plus vector (JV1139, open triangles), and ΔicmS ΔclpA::CmR plus vector (JV6296, open diamonds). Each time point represents the mean and standard deviation of colony forming units (CFU) recovered from triplicate wells. Growth curves are representative of three independent experiments.

(C) Protease susceptibility of DotL in the absence of IcmS and ClpA. L. pneumophila strains wild-type (Lp02) and ΔicmS ΔclpA::CmR (JV339) were grown to late stationary phase in broth, cells were sonicated, and treated with protease from Streptomyces griseus (Sigma) at the following concentrations: lane 1 (0 μM), lane 2 (5 μM), lane 3 (10 μM), lane 4 (20 μM), lane 5 (40 μM), lane 6 (80 μM), and lane 7 (160 μM). Samples were analyzed by Western blotting using antibodies specific to the proteins listed beneath each panel. Strains used are listed on the left. Results shown are representative of three independent experiments.