TheLegionella-driven PtdIns(4)Pgradient at LCV-ER membrane contact sites promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling

Abstract

AbstractThe causative agent of Legionnaires’ disease,Legionella pneumophila, governs interactions with host cells by secreting ca. 330 different “effector” proteins. The facultative intracellular bacteria replicate in macrophages and amoeba within a unique compartment, theLegionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide (PI) lipid conversion from PtdIns(3)Pto PtdIns(4)P, fusion with endoplasmic reticulum (ER)-derived vesicles and a tight association with the ER. Proteomics of purified LCVs revealed the presence of membrane contact sites (MCS) proteins implicated in lipid exchange. Using dually fluorescence-labeledDictyostelium discoideumamoeba, we reveal that the VAMP-associated protein (Vap), the PtdIns(4)P4-phosphatase Sac1, and the large fusion GTPase Sey1/atlastin-3 localize to the ER, but not to the LCV membrane, and that these ER-resident proteins promote intracellular replication ofL. pneumophilaand LCV remodeling. Moreover, oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and promote (OSBP8) or restrict (OSBP11) intracellular replication ofL. pneumophilaand LCV expansion. Furthermore, the PtdIns(4)P-subvertingL. pneumophilaeffectors LepB and SidC also promote LCV remodeling. Taken together, theLegionella- and host cell-driven PtdIns(4)Pgradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole remodeling.