Legionella pneumophila Effector Proteins

HiBiT-tagged effector proteins were overexpressed in L. pneumophila using the plasmid pxDC61. One day prior to infection, RAW 264.7 macrophages expressing LgBiT [28 (link),29 (link)] were seeded in a white clear-bottom 96 well cell culture plate (Greiner) at 8 x 10⁴ cells/well. L. pneumophila strains were grown for 2-3 days on BCYE-Agar plates supplemented with “BCYE Growth Supplements” (Oxoid) and appropriate antibiotics. Gene expression was induced by incubation of bacteria at 37 °C on BCYE agar plates with 0.5 mM IPTG for 24 hours. 100 μl of bacteria resuspended in HBSS + DrkBiT (1:1000) at 1.6x10⁸ cfu/ml were used to infect the RAW 264.7 macrophages (MOI = 200). After centrifugation at 300 x g for 8 minutes, 25 μl of NanoGlo® live cell buffer (Promega) supplemented with 1:20 of the extended live cell substrate Endurazine^™ (Promega) was added to each well. Luminescence was measured at 37°C and 5 % CO₂ every 15 minutes for 12 hours in a Tecan Spark plate reader.