Legionella -Containing Vacuoles Capture PtdIns(4) P -Rich Vesicles Derived from the Golgi Apparatus.

Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires' disease. The facultative intracellular bacterium employs the Icm/Dot type IV secretion system (T4SS) and a plethora of translocated "effector" proteins to interfere with host vesicle trafficking pathways and establish a replicative niche, the Legionella -containing vacuole (LCV). Internalization of the pathogen and the events immediately ensuing are accompanied by host cell-mediated phosphoinositide (PI) lipid changes and the Icm/Dot-controlled conversion of the LCV from a PtdIns(3) P -positive vacuole into a PtdIns(4) P -positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. The source and formation of PtdIns(4) P are ill-defined. Using dually labeled Dictyostelium discoideum amoebae and real-time high-resolution confocal laser scanning microscopy (CLSM), we show here that nascent LCVs continuously capture and accumulate PtdIns(4) P -positive vesicles from the host cell. Trafficking of these PtdIns(4) P -positive vesicles to LCVs occurs independently of the Icm/Dot system, but their sustained association requires a functional T4SS. During the infection, PtdIns(3) P -positive membranes become compacted and segregated from the LCV, and PtdIns(3) P -positive vesicles traffic to the LCV but do not fuse. Moreover, using eukaryotic and prokaryotic PtdIns(4) P probes (2×PHFAPP -green fluorescent protein [2×PHFAPP -GFP] and P4CSidC -GFP, respectively) along with Arf1-GFP, we show that PtdIns(4) P -rich membranes of the trans -Golgi network associate with the LCV. Intriguingly, the interaction dynamics of 2×PHFAPP -GFP and P4CSidC -GFP are spatially separable and reveal the specific PtdIns(4) P pool from which the LCV PI originates. These findings provide high-resolution real-time insights into how L. pneumophila exploits the cellular dynamics of membrane-bound PtdIns(4) P for LCV formation. IMPORTANCE The environmental bacterium Legionella pneumophila causes a life-threatening pneumonia termed Legionnaires' disease. The bacteria grow intracellularly in free-living amoebae as well as in respiratory tract macrophages. To this end, L. pneumophila forms a distinct membrane-bound compartment called the Legionella -containing vacuole (LCV). Phosphoinositide (PI) lipids are crucial regulators of the identity and dynamics of host cell organelles. The PI lipid PtdIns(4) P is a hallmark of the host cell secretory pathway, and decoration of LCVs with this PI is required for pathogen vacuole maturation. The source, dynamics, and mode of accumulation of PtdIns(4) P on LCVs are largely unknown. Using Dictyostelium amoebae producing different fluorescent probes as host cells, we show here that LCVs rapidly acquire PtdIns(4) P through the continuous interaction with PtdIns(4) P -positive host vesicles derived from the Golgi apparatus. Thus, the PI lipid pattern of the secretory pathway contributes to the formation of the replication-permissive pathogen compartment.