Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection

ABSTRACT The causative agent of Legionnaires ’ disease, Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector ” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba Dictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficient L. pneumophila, PtdIns(3,4,5)P3 transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)P within 1 min after uptake. Whereas phagosomes containingicmT mutant bacteria remained decorated with PtdIns(3)P, more than 80 % of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major re-arrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)P transiently localized to early phago-somes harboring wild-type oricmT L. pneumophila and was cleared within minutes after uptake. During the following 2 h, PtdIns(4)P steadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)P identity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboringsidC-sdcA mutant bacteria defective for ER recruitment, without affecting ini-tial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids impli-cated in LCV formation and provide insight into host cell membrane and effector protein interactions