Temporal regulation of SidH is mediated by ubiquitin ligase LubX and host proteasome.

(A) Time course of intracellular SidH and LubX levels after infection with L. pneumophila strain producing the triple FLAG-tagged SidH. CHO-FcγRII cells were infected and digitonin extracts were prepared at indicated time after infection as in Figure 3B. The Ni lane denotes the non-infected control. Immunoprecipitation and immunoblotting was carried out as in Figure 3B. The asterisks denote a non-specific signal. (B) Shutdown of SidH requires ubiquitin ligase activity of LubX. CHO-FcγRII cells infected with L. pneumophila strains carrying wild-type lubX gene or lubXI39A U-box1 dead mutant were analyzed as in panel A. (C) Shutdown of SidH requires host proteasome. CHO-FcγRII cells were treated with 10 µM MG132 or 0.1% DMSO from 30 minutes before infection with L. pneumophila. (D) Shutdown of SidH requires bacterial protein synthesis after infection. L. pneumophila was pretreated with 100 µg/ml gentamicin (Gm) or water for 30 minutes and used for infection. (E) Delayed delivery of LubX results in proteasomal degradation of SidH at later stages of infection.