The Mycobacterium bovis bacille Calmette-Guerin phagosome proteome.
Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin (BCG) alter the maturation of their phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified phagosomes from BCG-infected human macrophages and analyzed the phagosomes by Western immunoblotting and mass spectrometry-based proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP-2, Niemann-Pick protein C1, and syntaxin 3 were readily detectable on the BCG phagosome but at levels that were lower than on the latex bead phagosome; flotillin-1 and the vacuolar ATPase were barely detectable on the BCG phagosome but highly enriched on the latex bead phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG phagosomes, and a partially overlapping set of 289 human proteins on latex bead phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG phagosome preparations were also identified on latex bead phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG phagosomes that were not identified in any of our latex bead phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPase-activating-like protein IQGAP1, and proteins of unknown function, such as FAM3C. Our phagosome purification procedure and initial proteomics analyses set the stage for a quantitative comparative analysis of mycobacterial and latex bead phagosome proteomes.
Lee
Bai-Yu
BY
Division of Infectious Diseases, Department of Medicine, Center for Health Sciences, University of California-Los Angeles School of Medicine, Los Angeles, California 90095-1688, USA.
Jethwaney
Deepa
D
Schilling
Birgit
B
Clemens
Daniel L
DL
Gibson
Bradford W
BW
Horwitz
Marcus A
MA
D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000378:metabolism / Q000382:microbiology; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000077296:Niemann-Pick C1 Protein; D010588:Phagosomes / Q000378:metabolism* / Q000382:microbiology / Q000648:ultrastructure; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D050765:Qa-SNARE Proteins / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D020543:Proteome; D050765:Qa-SNARE Proteins; C106354:flotillins; D049452:Green Fluorescent Proteins; D025262:Vacuolar Proton-Translocating ATPases