Infecting Cells with Salmonella and Legionella

S. Typhimurium SR-11 x3181 carrying an mCherry-expressing plasmid (Hubber et al., 2014 (link)) was used for infection throughout the study. Legionella strains used in this study were derivatives of L. pneumophila strains Philadelphia-1 (Lp01) (Berger and Isberg, 1993 (link)). Salmonella was grown in Luria-Bertani (LB) medium containing 20 μg/ml of chloramphenicol to maintain the mCherry expressing plasmid. Legionella strains were grown in liquid N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffered yeast extract (AYE) media (Horwitz and Silverstein, 1983 (link)). The ravZ and dotA deletion strains were constructed by allelic exchange as described previously (Zuckman et al., 1999 (link)). Eukaryotic expression plasmids for RavZ were constructed by cloning the PCR-amplified wild-type or mutant (C258A) ravZ into the p3xFLAG-CMV-10 expression vector (Sigma). Site-directed mutagenesis was conducted using the Quickchange II Site-Directed Mutagenesis Kit according to the manufacturer's recommendations (Agilent Technologies).