Lem8 Translocation Assay in L. pneumophila

To test the Dot/Icm-dependent translocation into host cells, Lem8 was cloned intro pXDC61m (Zhu et al., 2011 (link)) to generate s β-lactamase-Lem8 fusion. This plasmid was introduced wild-type or the dotA⁻ mutant of L. pneumophila and the resulting strains were used to infect U937 cells at an multiplicity of infection (MOI) of 20 after 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) induction. One hour after infection, the CCF4-AM substrates (Invitrogen, Carlsbad, CA) were added into the medium and the cells were incubated for another 2 hr at 25°C, followed by image acquisition using an Olympus IX-83 fluorescence microscope. The translocation of Lem8 was assessed by calculating the percentage of cells emitting blue fluorescence.