2014
DOI: 10.1021/pr500765x
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Complete and Ubiquitinated Proteome of theLegionella-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 lysine48-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 purif… Show more

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Cited by 35 publications
(55 citation statements)
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References 80 publications
(246 reference statements)
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“…These host vesicles are thought to deliver membrane material, foodstuffs, and other "cargo" that facilitate intravacuolar growth of L. pneumophila (86). Among the host GTPases that modulate ER-to-Golgi vesicle traffic, Rab1B is well known for being recruited rapidly and substantially to LCVs in A/J BMD macrophages and U937 cells as well as murine RAW264.7 macrophages, human A549 lung epithelial cells, and human HEK293 embryonic kidney cells (61,66,67,(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98). Expression of a dominant negative form of Rab1 restricts L. pneumophila growth in Chinese hamster ovary (CHO) cells and COS1 cells, suggesting that Rab1B recruitment enhances bacterial growth (61,66).…”
Section: Bmdm-ltmentioning
confidence: 99%
See 1 more Smart Citation
“…These host vesicles are thought to deliver membrane material, foodstuffs, and other "cargo" that facilitate intravacuolar growth of L. pneumophila (86). Among the host GTPases that modulate ER-to-Golgi vesicle traffic, Rab1B is well known for being recruited rapidly and substantially to LCVs in A/J BMD macrophages and U937 cells as well as murine RAW264.7 macrophages, human A549 lung epithelial cells, and human HEK293 embryonic kidney cells (61,66,67,(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98). Expression of a dominant negative form of Rab1 restricts L. pneumophila growth in Chinese hamster ovary (CHO) cells and COS1 cells, suggesting that Rab1B recruitment enhances bacterial growth (61,66).…”
Section: Bmdm-ltmentioning
confidence: 99%
“…As noted above, at least 15 studies have documented the association between Rab1B and LCVs; however, only 3 of these studies used human macrophages, and in no case was there a direct comparison between murine and human host cells (66,93,97). Therefore, we infected U937 cells with WT 130b and its lspF mutant and monitored the association of Rab1B with the LCVs, using the same immunofluorescence assay as that used to assess BMD mac- rophages.…”
Section: Bmdm-ltmentioning
confidence: 99%
“…Since many host SLC amino acid transporters have been detected in the proteome of the LCV within macrophages (7)(8)(9), it is likely that import of amino acids is mediated by some of the SLCs that are incorporated into the LCV membrane. The SLC1A5 neutral amino acid transporter has been shown to be required for intravacuolar growth of L. pneumophila (55), but its potential LCV localization remains to be validated.…”
Section: Discussionmentioning
confidence: 99%
“…Upon entry into human alveolar macrophages, L. pneumophila is localized within a vacuole designated the Legionella-containing vacuole (LCV), which evades the endocytic pathway and is rapidly remodeled by the rough endoplasmic reticulum (5,6). High-throughput analyses of the proteome of the LCV have shown selective acquisition of numerous host proteins (7)(8)(9). Remodeling of the LCV is mediated by ϳ300 bacterial effector proteins that are injected into the host cell via the Dot/Icm type IVb secretion system (5,(10)(11)(12).…”
mentioning
confidence: 99%
“…For all three bacterial species tested, the SP3* protocol enabled an increased peptide and protein identification rate compared to the CD+ISD protocol ( Figure ; Figure S1 and , Supporting Information). For L. pneumophila to our knowledge only global proteome data obtained with bacterial numbers at least 50 times larger than those from infection settings, are currently available . In the present work, on average 835 peptides that represented 303 proteins or 9% of the theoretical proteome could be identified with CD+ISD from 2 × 10 6 L. pneumophila bacteria.…”
mentioning
confidence: 83%