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.
Key wordsAtlastin Host-pathogen interactions Intracellular bacteria Large GTPase Legionella pneumophila Macrophage Epithelial cells Pathogen vacuole Type IV secretion RNA interference
ADP-ribosylation factor 1
Green fluorescent protein
Heat shock protein 90
Intracellular multiplication/defective organelle trafficking
Multiplicity of infection
RNA-induced silencing complex
Small interfering RNA
Type IV secretion system
We would like to thank Stephen Weber and Daniel Strebinger for providing critical input on the manuscript. Work in the group of H.H. was supported by the Swiss National Science Foundation (SNF; 31003A_153200), the Novartis Foundation for Medical-Biological Research, and the OPO Foundation.