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Purification of Pathogen Vacuoles from Legionella-infected Phagocytes
Published on: June 19, 2012
Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages
William M Bruckert1, Yousef Abu Kwaik
1Department of Microbiology and Immunology, University of Louisville , 319 Abraham Flexner Way 55A, Louisville, Kentucky 40202, United States.
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.
Insights
Legionella pneumophila uses the AnkB effector to decorate its vacuole with ubiquitinated proteins, essential for replication. This study identifies the host proteins within these vacuoles, revealing complex pathogen-host interactions during infection.
Area of Science:
- Microbiology
- Cell Biology
- Proteomics
Background:
- Legionella pneumophila evades host defenses by replicating within a specialized vacuole (LCV).
- The AnkB effector protein is crucial for decorating the LCV with ubiquitinated proteins, facilitating bacterial replication.
- Understanding the host proteome within the LCV is key to deciphering L. pneumophila pathogenesis.
Purpose of the Study:
- To identify and characterize the host-derived proteome, including ubiquitinated proteins, associated with L. pneumophila LCVs.
- To compare the proteomes of LCVs from wild-type (WT) and AnkB-mutant strains of L. pneumophila.
- To elucidate the role of ubiquitination and AnkB in LCV biogenesis and host-pathogen interactions.
Main Methods:
- High-throughput liquid chromatography-tandem mass spectrometry (LC-MS) was employed.
- Purification of LCVs from human U937 macrophages infected with WT and AnkB mutant L. pneumophila strains.
- Bioinformatic analysis of identified host proteins and ubiquitinated proteins, including pathway enrichment analysis.
Main Results:
- LCVs from WT strains contained 1193 proteins (24 ubiquitinated), while AnkB mutant LCVs contained 1546 proteins (29 ubiquitinated).
- Both WT and mutant LCVs showed enrichment of proteins involved in signaling, protein transport, and metabolism.
- AnkB mutant LCVs were enriched for proteins related to transcription/translation and immune responses, while ubiquitinated proteins on WT LCVs were linked to immune response and intracellular trafficking.
Conclusions:
- The study provides a comprehensive proteomic landscape of the LCV within human macrophages.
- The findings highlight the complex and dynamic nature of LCV biogenesis and the significant role of AnkB-mediated ubiquitination.
- This research offers a valuable resource for future investigations into Legionella pathogenesis and host-pathogen interactions.
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