RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42

The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G‐nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome‐scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE‐mediated entry. Follow‐up assays assigned these ‘hits’ to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella ‐containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI‐facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs. Synopsis Pathogens are not only a menace to public health, but they also provide excellent tools for probing host cell function. Thus, studying infection mechanisms has fueled progress in cell biology (Ridley et al , 1992 ; Welch et al , 1997 ). In the presented study, we have performed an RNAi screen to identify host cell genes required for Salmonella host cell invasion. This screen identified proteins known to contribute to Salmonella ‐induced actin rearrangements (e.g., Cdc42 and the Arp2/3 complex; reviewed in Schlumberger and Hardt, 2006 ) and vesicular traffic (e.g., Rab7) as well as unexpected hits, such as the COPI complex. COPI is a known organizer of Golgi‐to‐ER vesicle transport (Bethune et al , 2006 ; Beck et al , 2009 ). Here, we show that COPI is also involved in plasma membrane targeting of cholesterol, sphingolipids and the Rho GTPases Cdc42 and Rac1, essential host cell factors required for Salmonella invasion. This explains why COPI depletion inhibits infection by S . Typhimurium and illustrates how combining bacterial pathogenesis and systems approaches can promote cell biology. Salmonella Typhimurium is a common food‐borne pathogen and worldwide a major public health problem causing severe diarrhea. The