Figure 4. Targeting of DrrA by PI4P-binding is functionally important during L. pneumophila infection because it is required for localization of DrrA and Rab1 to Legionella-containing vacuoles.

(A) Micrographs of HEK293 FcγR cells infected with various Legionella pneumophila Philadelphia-1 strains at 1 hour post-infection stained with Rab1b-specific antibodies and DAPI. Strains that do not produce DrrA (ΔdrrA) or produce DrrA containing mutations that perturb PI4P-binding (ΔdrrA +pJBdrrA K568A/T619A) fail to show vacuolar Rab1b signals. (B) Quantification of Rab1b recruitment to LCVs with wild type, mutant and complemented L. pneumophila strains at various time points post-infection. Vacuoles were stained for endogenous Rab1 recruitment using Rab1b specific antibody. At each time point 150 vacuoles were counted per strain from three independent samples. Data shown are from one experiment but in at least two other independent experiments the same Rab1b recruitment phenotypes were observed. Numbers shown above bars indicate percent infected.