L. pneumophila governs by means of the Icm/Dot T4SS and more than 150 effector proteins the 1) uptake, 2) inhibition of endocytosis, 3) interactions with the early secretory vesicle trafficking pathway and the ER, 4) intracellular replication and 5) release from amoebae. Formation of the Legionella-containing vacuole (LCV) involves small GTPases and phosphoinositide (PI) lipids of the host cell. Phosphoinositides are key regulators of signal transduction and membrane dynamics in eukaryotic cells. By using D. discoideum mutant strains, we found that PI 3-kinases and a PI 5-phosphatase (Dd5P4) inhibit intracellular replication of L. pneumophila. Dd5P4 is a homologue of human OCRL1 (Oculocerebrorenal syndrome of Lowe), implicated in retrograde vesicle trafficking. Moreover, we identified phosphatidylinositol-4 phosphate (PtdIns(4)P) on LCVs and Icm/Dot-secreted proteins binding to specific PIs in vitro. These effector proteins include LpnE, which interacts with OCRL1, the Rab 1 guanine nucleotide exchange factor SidM, and SidC, which recruits ER to replication-permissive LCVs. Thus, L. pneumophila exploits host cell PIs to anchor Icm/Dot-translocated effector proteins to the LCV membrane.

Intact LCVs can be enriched in a simple two-step process from L. pneumophila-infected D. discoideum producing the ER/LCV marker GFP-calnexin. The protocol includes immuno-magnetic separation by magnetic beads, using an antibody against an L. pneumophila effector protein exclusively localizing to the LCV membrane, followed by density gradient centrifugation. We determined by LC-MS/MS the proteome of LCVs at different time points post infection. Among more than 560 proteins identified were several small GTPases and other host proteins previously not implicated in LCV formation. The functional relevance of some of the identified proteins was verified by co-localization studies and RNA interference.