Perturbation of Legionella Cell Infection by RNA Interference

Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.