Legionella pneumophila Exploits PI(4)P to Anchor Secreted Effector Proteins to the Replicative Vacuole

Abstract

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot–secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV. The bacterium Legionella pneumophila causes Legionnaires' disease, a severe pneumonia. In the environment, L. pneumophila multiplies within amoebae. By inhaling contaminated water droplets, L. pneumophila is transmitted to the human lung and grows within immune cells (macrophages). Within amoebae and macrophages, L. pneumophila replicates with a similar mechanism by forming a membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of the LCV is not well defined, but requires that L. pneumophila injects proteins into the host cell via the intracellular multiplication (Icm)/defective organelle trafficking (Dot) secretion system. Phosphoinositide (PI) lipids are central mediators of membrane dynamics in amoebae and mammalian cells. In this study, the researchers report that phosphatidylinositol(3) kinases (enzymes that add a phosphate residue to PIs) are dispensable for uptake of L. pneumophila, but affect the formation of the LCV, as well as uptake and degradation of mutant L. pneumophila lacking a functional Icm/Dot system. Icm/Dot–secreted proteins were identified which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P), a marker of the Golgi organelle in the secretory pathway, which is also present on the LCV. These findings indicate that L. pneumophila exploits PI(4)P to anchor secreted effector proteins to the LCV, thus subverting host cell PI metabolism to establish its replicative niche.