Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling
Abstract
Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.
Synopsis
Legionella pneumophila replicates intracellularly within a unique “Legionella-containing vacuole” (LCV). Dually fluorescence-labeled Dictyostelium discoideum amoeba reveal that LCVs and the ER form membrane contact sites and Legionella- as well as host cell-driven lipid flux promotes pathogen vacuole remodeling.
- The Legionella-containing vacuole (LCV) engages in membrane contact sites (MCS) with the ER.
- The MCS components VAMP-associated protein (Vap), oxysterol binding proteins (OSBPs), and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER and/or the LCV membrane.
- Host cell factors (Vap, OSBPs, Sac1) as well as bacterial effector proteins (LepB, SidC) modulate lipid exchange, LCV remodelling and intracellular growth of L. pneumophila.
