Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages

Abstract

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.

Keywords: AnkB; Dot/Icm; LCV; Legionnaires’ disease; polyubiquitin; proteome; signaling.

Figure 1

LCV purification using a discontinuous sucrose gradient. U937 macrophages were infected with WT L. pneumophila or the isogenic ankB strain at an MOI of 50 for 30 min washed and the infection proceeded for 4 h. Cells were lysed through dounce homogenization, and the postnuclear supernatant was used to isolate LCVs through density ultracentrifugation on a discontinuous sucrose gradient. (A) Diagram of the sucrose gradient showing the isolated LCVs at 55–65% interface and (B) confocal microscopy of isolated LCVs labeled with mouse anti-L. pneumophila following vacuole membrane permeabilization. (C) Confocal microscopy of isolated LCVs labeled with rabbit anti-L. pneumophila antiserum and mouse antipolyubiquitin antibodies.

Figure 2

Functional classification of eukaryotic proteins localized to the LCV. The 1193 eukaryotic proteins localized to the WT strain LCV and the 1546 localized to the ankB mutant LCV identified by high-throughput LC–MS were grouped according to their cellular function according to the UniProt and GeneCards database sets.

Figure 3

MetaCore enrichment analysis of metabolic networks in the WT STRAIN LCV proteome. The complete WT strain LCV proteome was analyzed through MetaCore software, using the enrichment analysis function, to identify the most significant metabolic networks.