Legionella-Containing Vacuoles Capture PtdIns(4)-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