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 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.

A Type IV Translocated Legionella Cysteine Phytase Counteracts Intracellular Growth Restriction by Phytate

“…In support of this notion, L. pneumophila itself is taken up by phagocytes by macropinocytic rather than phagocytic processes (21,55). Thus, the exogenously added phytate might reach the pathogen compartment through vesicle fusion, yet transmembrane transport processes might also play a role.…”

“…Finally, the observation of PtdIns(4)P in D. discoideum by GFP-SidC P4C was carried out as described previously (21).…”

“…Thus, we hypothesized that LppA might also modulate the LCV PI pattern in L. pneumophila-infected cells. To compare the dynamics of PtdIns(4)P accumulation on LCVs harboring wild type or ⌬lppA mutant bacteria, we performed live cell imaging using specific PI probes (21). Upon infection of D. discoideum amoebae producing GFP-SidC P4C with L. pneumophila, LCVs harboring wild type or ⌬lppA bacteria accumulated PtdIns(4)P to the same extent within 1 or 2 h postinfection (Fig.…”

“…Some L. pneumophila Icm/Dot substrates target and subvert pivotal regulators of eukaryotic signal transduction and vesicle trafficking, such as small GTPases (8 -13) or phosphoinositide (PI) lipids (14 -16). Several Legionella effectors anchor to the LCV membrane by specifically binding to the phosphorylated phosphatidylinositol (PtdIns) headgroup of PI lipids, namely PtdIns(4)P (17)(18)(19)(20)(21)(22) and PtdIns(3)P (23)(24)(25), which are implicated in secretory and endosomal vesicle trafficking, respectively. Moreover, L. pneumophila produces two non-homologous Icm/Dot-translocated PI 3-phosphatases, SidF and SidP, which might modulate the LCV PI pattern (26,27).…”

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

“…Intravacuolar pathogens subvert PI metabolism to manipulate the lipid profile of their host-derived replicative niche, thus controlling the interactions with the endosomal maturation pathway according to their needs. Mycobacterium tuberculosis (35,36) and Legionella pneumophila (37,38) use effectors to deplete PI(3)P at the surface of their vacuoles to block phagosomal maturation and avoid fusion with degradative compartments. On the contrary, Salmonella enterica serovar Typhimurium uses the SPI-1 substrate SopB to maintain elevated levels of PI(3)P at the surface of Salmonella-containing vacuoles, favoring their biogenesis (39)(40)(41).…”