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 containing ΔicmT 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 rearrangement of PtdIns(3)P-positive membranes condensing to the cell center.
PtdIns(4)P transiently localized to early phagosomes harboring wild-type or ΔicmT 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 harboring ΔsidC-sdcA mutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI
lipids implicated in LCV formation and provide insight into host cell membrane and effector
protein interactions.
IMPORTANCE: The environmental bacterium Legionella pneumophila is the causative agent of Legionnaires'
pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, the Legionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different
proteins into host cells. Some of these
proteins bind
phosphoinositide (PI)
lipids to decorate the LCV. PI
lipids are crucial factors involved in host cell membrane dynamics and LCV formation. Using Dictyostelium amoebae producing one or two distinct
fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI
lipid PtdIns(3)P was slowly cleared from LCVs, thus incapacitating the host cell's digestive machinery, while
PtdIns(4)P gradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector
proteins and therefore represents a crucial aspect of LCV formation.