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

Bruckert WM, Abu Kwaik Y - J. Proteome Res. (2014)

Bottom Line: 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.The ankB mutant LCVs are preferentially enriched for proteins involved in transcription/translation and immune responses.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

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LCV purification usinga discontinuous sucrose gradient. U937 macrophageswere infected with WT L. pneumophila or the isogenic ankB strain at an MOI of 50 for 30 min washed and the infectionproceeded for 4 h. Cells were lysed through dounce homogenization,and the postnuclear supernatant was used to isolate LCVs through densityultracentrifugation on a discontinuous sucrose gradient. (A) Diagramof the sucrose gradient showing the isolated LCVs at 55–65%interface and (B) confocal microscopy of isolated LCVs labeled withmouse anti-L. pneumophila following vacuole membranepermeabilization. (C) Confocal microscopy of isolated LCVs labeledwith rabbit anti-L. pneumophila antiserum and mouseantipolyubiquitin antibodies.
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fig1: LCV purification usinga discontinuous sucrose gradient. U937 macrophageswere infected with WT L. pneumophila or the isogenic ankB strain at an MOI of 50 for 30 min washed and the infectionproceeded for 4 h. Cells were lysed through dounce homogenization,and the postnuclear supernatant was used to isolate LCVs through densityultracentrifugation on a discontinuous sucrose gradient. (A) Diagramof the sucrose gradient showing the isolated LCVs at 55–65%interface and (B) confocal microscopy of isolated LCVs labeled withmouse anti-L. pneumophila following vacuole membranepermeabilization. (C) Confocal microscopy of isolated LCVs labeledwith rabbit anti-L. pneumophila antiserum and mouseantipolyubiquitin antibodies.

Mentions: To determinethe identity of host proteins on the LCV, we infected U937 human macrophageswith L. pneumophila WT strain AA100/130b or its isogenic ankB mutant strain. The ankB mutant strainevades lysosomal fusion and is localized within an ER-derived LCV,similar to the WT strain.16 However, the ankB mutant strain fails to replicate within human macrophagesand amoeba due to the levels of cellular amino acids being below thethreshold needed as the major source of carbon and energy to supportintravacuolar proliferation of L. pneumophila.13,14,43 The U937 human macrophage cellline is widely used in studies on Legionella-humanmacrophage interaction and was used in this study instead of primaryhuman monocytes due to the need of a large number of cells to isolateLCVs, because only ∼20% of the cells become infected. Followinga 4 h infection of 0.6 × 109 U937 macrophages by eachof the strains, the LCVs were purified according to previously reportedprotocols with minor modifications.25,44 Cells werelysed through dounce homogenization, and the PNS was used to isolateLCVs through density ultracentrifugation on a discontinuous sucrosegradient, which resulted in purified LCVs at the 55–65% interface(Figure 1A). To ensure vacuole integrity followingpurification, isolated LCVs were evaluated using confocal microscopyafter differential membrane permeabilization as well as vacuole markerstaining to ensure the LCV membranes were intact. Vacuoles were labeledwith a polyclonal anti-Legionella antibody priorto vacuolar permeabilization, which resulted in ∼20% of bacteriabeing labeled, while 100% of bacteria were labeled after vacuolarmembrane permeabilization, indicating that the LCV membrane is intacton ∼80% of the isolated LCVs (Figure 1B). We next evaluated the presence of polyubiquitinated proteins,which showed that ∼70% of isolated LCVs were decorated withpolyubiqutinated proteins (Figure 1C). IsolatedLCVs were solubilized in 1% Triton X-100, and the eukaryotic proteinsassociated with the LCV were identified by high-throughput liquidchromatography coupled to tandem mass spectrometry (LC–MS).The MS was loaded with 2 μg of protein for each LCV sample.A positive protein identification for the proteome was based on atleast two unique peptides. Although numerous proteins were identified,it is likely that some proteins present with scarce quantities onthe LCV are not detectable by our analyses.


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

Bruckert WM, Abu Kwaik Y - J. Proteome Res. (2014)

LCV purification usinga discontinuous sucrose gradient. U937 macrophageswere infected with WT L. pneumophila or the isogenic ankB strain at an MOI of 50 for 30 min washed and the infectionproceeded for 4 h. Cells were lysed through dounce homogenization,and the postnuclear supernatant was used to isolate LCVs through densityultracentrifugation on a discontinuous sucrose gradient. (A) Diagramof the sucrose gradient showing the isolated LCVs at 55–65%interface and (B) confocal microscopy of isolated LCVs labeled withmouse anti-L. pneumophila following vacuole membranepermeabilization. (C) Confocal microscopy of isolated LCVs labeledwith rabbit anti-L. pneumophila antiserum and mouseantipolyubiquitin antibodies.
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Related In: Results  -  Collection

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fig1: LCV purification usinga discontinuous sucrose gradient. U937 macrophageswere infected with WT L. pneumophila or the isogenic ankB strain at an MOI of 50 for 30 min washed and the infectionproceeded for 4 h. Cells were lysed through dounce homogenization,and the postnuclear supernatant was used to isolate LCVs through densityultracentrifugation on a discontinuous sucrose gradient. (A) Diagramof the sucrose gradient showing the isolated LCVs at 55–65%interface and (B) confocal microscopy of isolated LCVs labeled withmouse anti-L. pneumophila following vacuole membranepermeabilization. (C) Confocal microscopy of isolated LCVs labeledwith rabbit anti-L. pneumophila antiserum and mouseantipolyubiquitin antibodies.
Mentions: To determinethe identity of host proteins on the LCV, we infected U937 human macrophageswith L. pneumophila WT strain AA100/130b or its isogenic ankB mutant strain. The ankB mutant strainevades lysosomal fusion and is localized within an ER-derived LCV,similar to the WT strain.16 However, the ankB mutant strain fails to replicate within human macrophagesand amoeba due to the levels of cellular amino acids being below thethreshold needed as the major source of carbon and energy to supportintravacuolar proliferation of L. pneumophila.13,14,43 The U937 human macrophage cellline is widely used in studies on Legionella-humanmacrophage interaction and was used in this study instead of primaryhuman monocytes due to the need of a large number of cells to isolateLCVs, because only ∼20% of the cells become infected. Followinga 4 h infection of 0.6 × 109 U937 macrophages by eachof the strains, the LCVs were purified according to previously reportedprotocols with minor modifications.25,44 Cells werelysed through dounce homogenization, and the PNS was used to isolateLCVs through density ultracentrifugation on a discontinuous sucrosegradient, which resulted in purified LCVs at the 55–65% interface(Figure 1A). To ensure vacuole integrity followingpurification, isolated LCVs were evaluated using confocal microscopyafter differential membrane permeabilization as well as vacuole markerstaining to ensure the LCV membranes were intact. Vacuoles were labeledwith a polyclonal anti-Legionella antibody priorto vacuolar permeabilization, which resulted in ∼20% of bacteriabeing labeled, while 100% of bacteria were labeled after vacuolarmembrane permeabilization, indicating that the LCV membrane is intacton ∼80% of the isolated LCVs (Figure 1B). We next evaluated the presence of polyubiquitinated proteins,which showed that ∼70% of isolated LCVs were decorated withpolyubiqutinated proteins (Figure 1C). IsolatedLCVs were solubilized in 1% Triton X-100, and the eukaryotic proteinsassociated with the LCV were identified by high-throughput liquidchromatography coupled to tandem mass spectrometry (LC–MS).The MS was loaded with 2 μg of protein for each LCV sample.A positive protein identification for the proteome was based on atleast two unique peptides. Although numerous proteins were identified,it is likely that some proteins present with scarce quantities onthe LCV are not detectable by our analyses.

Bottom Line: 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.The ankB mutant LCVs are preferentially enriched for proteins involved in transcription/translation and immune responses.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

Show MeSH
Related in: MedlinePlus