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The phagosome proteome: insight into phagosome functions.

ABSTRACT: Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.

SUBMITTER: Garin J

PROVIDER: S-EPMC2193653 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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The phagosome proteome: insight into phagosome functions.

Garin J J Diez R R Kieffer S S Dermine J F JF Duclos S S Gagnon E E Sadoul R R Rondeau C C Desjardins M M

The Journal of cell biology 20010101 1

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating ou ...[more]

PMID: 11149929

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Project description: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.

Project description:The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions. All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 Âµm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record. http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

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The phagosome proteome: insight into phagosome functions.

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The phagosome proteome: insight into phagosome functions.

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