Legionella-Containing Vacuoles Capture Ptdlns(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 lcm/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 lcm/Dot-controlled conversion of the LCV from a Ptdlns(3)Ppositive vacuole into a Ptdlns(4)P-positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. The source and formation of Ptdlns(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 Ptdlns(4)P-positive vesicles from the host cell. Trafficking of these Ptclins(4)P-positive vesicles to LCVs occurs independently of the lcm/Dot system, but their sustained association requires a functional T4SS. During the infection, Ptdlns(3)P-positive membranes become compacted and segregated from the LCV, and Ptdlns(3)P-positive vesicles traffic to the LCV but do not fuse. Moreover, using eukaryotic and prokaryotic Ptclins(4)P probes (2xPH(FAPP)-green fluorescent protein [2xPH(FAPP)-GFP] and P4C(Sidc)-GFP, respectively) along with Arfl-GFP, we show that Ptdlns(4)P-rich membranes of the trans-Golgi network associate with the LCV. Intriguingly, the interaction dynamics of 2xPH(FAPP)-GFP and P4C(Sidc)-GFP are spatially separable and reveal the specific Ptdlns(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 Ptdlns(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 Ptdlns(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 Ptclins(4)P on LCVs are largely unknown. Using Dictyostelium amoebae producing different fluorescent probes as host cells, we show here that LCVs rapidly acquire Ptdlns(4)P through the continuous interaction with Ptdlns(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.