Complete and Ubiquitinated Proteome of the Legionella-Containing Vacuole within Human Macrophages

[Image: see text] Within protozoa or human macrophages Legionella pneumophila evades the endosomal pathway and replicates within an ER-derived vacuole termed the Legionella-containing vacuole (LCV). The LCV membrane-localized AnkB effector of L. pneumophila is an F-box protein that mediates decoration of the LCV with lysine(48)-linked polyubiquitinated proteins, which is essential for intravacuolar replication. Using high-throughput LC–MS analysis, we have identified the total and ubiquitinated host-derived proteome of LCVs purified from human U937 macrophages. The LCVs harboring the AA100/130b WT strain contain 1193 proteins including 24 ubiquitinated proteins, while the ankB mutant LCVs contain 1546 proteins with 29 ubiquitinated proteins. Pathway analyses reveal the enrichment of proteins involved in signaling, protein transport, phosphatidylinositol, and carbohydrate metabolism on both WT and ankB mutant LCVs. The ankB mutant LCVs are preferentially enriched for proteins involved in transcription/translation and immune responses. Ubiquitinated proteins on the WT strain LCVs are enriched for immune response, signaling, regulation, intracellular trafficking, and amino acid transport pathways, while ubiquitinated proteins on the ankB mutant LCVs are enriched for vesicle trafficking, signaling, and ubiquitination pathways. The complete and ubiquitinated LCV proteome within human macrophages illustrates complex and dynamic biogenesis of the LCV and provides a rich resource for future studies.