Fig 2.

PikA is a substrate of Type IV secrete system. (A) HeLa cells were incubated with R. rickettsii (MOI = 1) for the indicated durations at 33°C. The presence of PikA in cell lysates was detected using antibodies specific for PikA, and actin was probed as a loading control. (B) HeLa cells infected with R. rickettsii (MOI = 1) for 0–4 days post-infection (D P.I.) were lysed with 0.1% Triton X-100, and lysates were fractionated by 10,000 g centrifugation. Uninfected cells were similarly processed as controls. Proteins in pellet and supernatant fractions were probed for PikA, the bacterial protein EF-Ts, and host protein GAPDH by immunoblotting. Similar results were obtained in three independent experiments. (C) Interactions between PikA and RvhD4 by a bacterial two-hybrid assay. The T25-PikA and T18-RvhD4 fusion were co-expressed in the reporter E. coli strain BTH101, strains harboring relevant combinations of plasmids were established as controls. Bacterial cells were steaked on LB plate containing X-Gal. Images were acquired after incubation at 37°C for 14 h. Note that strains capable of hydrolyzing X-Gal to form blue cells indicate positive interactions. (D and E) PikA is translocated into host cells by the L. pneumophila Dot/Icm transporter. Expression of the TEM1-PikA fusion. Cultures of L. pneumophila strains harboring the indicated plasmids were induced with 50 µM IPTG for 4 h at 37°C, and the fusion protein was probed by immunoblotting using anti-TEM antibodies. The metabolic enzyme isocitrate dehydrogenase (ICDH) was detected as a loading control. (D) Raw264.7 cells were challenged with Lp02 or Lp03 expressing the TEM1-PikA fusion at an MOI of 50 for 3 h prior to adding the CCF2-AM substrate. Samples incubated for an additional 2 h were used for image acquisition (E, top). Bar, 50 µm. Quantitation of cells emitting blue fluorescence signals. At least 300 cells were scored for each sample, and the results (mean ± s.e.) shown were from three independent experiments (E, bottom). ***, P < 0.001.