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The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome*

  • Bai-Yu Lee
    Affiliations
    Division of Infectious Diseases, Department of Medicine, Center for Health Sciences, University of California-Los Angeles School of Medicine, Los Angeles, California 90095-1688,
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  • Deepa Jethwaney
    Footnotes
    Affiliations
    Buck Institute for Age Research, Novato, California 94945, and
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  • Birgit Schilling
    Affiliations
    Buck Institute for Age Research, Novato, California 94945, and
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  • Daniel L. Clemens
    Correspondence
    To whom correspondence should be addressed: Division of Infectious Diseases, Dept. of Medicine, UCLA School of Medicine, CHS 37-121, 10833 LeConte Ave., Los Angeles, CA 90095-1688. Tel.: 310-825-9324; Fax: 310-794-7156;
    Affiliations
    Division of Infectious Diseases, Department of Medicine, Center for Health Sciences, University of California-Los Angeles School of Medicine, Los Angeles, California 90095-1688,
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  • Bradford W. Gibson
    Affiliations
    Buck Institute for Age Research, Novato, California 94945, and

    Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143
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  • Marcus A. Horwitz
    Affiliations
    Division of Infectious Diseases, Department of Medicine, Center for Health Sciences, University of California-Los Angeles School of Medicine, Los Angeles, California 90095-1688,
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants HL077000 and AI065359.
    The on-line version of this article (available at http://www. mcponline.org) contains supplemental Figs. 1–4 and Tables 1–12.
    ¶ Present address: Bio-Rad Laboratories, 825 Alfred Noble Dr., Hercules, CA 94547.
Open AccessPublished:October 07, 2009DOI:https://doi.org/10.1074/mcp.M900396-MCP200
      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.
      Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a facultative intracellular bacterium. In human macrophages, M. tuberculosis resides in a membrane-bound phagosomal compartment that resists fusion with lysosomes and is only mildly acidified (
      • Xu S.
      • Cooper A.
      • Sturgill-Koszycki S.
      • van Heyningen T.
      • Chatterjee D.
      • Orme I.
      • Allen P.
      • Russell D.G.
      Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages.
      ,
      • Malik Z.A.
      • Denning G.M.
      • Kusner D.J.
      Inhibition of Ca(2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.
      ,
      • Goren M.B.
      • D'Arcy Hart P.
      • Young M.R.
      • Armstrong J.A.
      Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis..
      ,
      • Crowle A.J.
      • Dahl R.
      • Ross E.
      • May M.H.
      Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic.
      ,
      • Armstrong J.A.
      • Hart P.D.
      Response of cultured macrophages to Mycobacterium tuberculosis with observations on fusion of lysosomes with phagosomes.
      ). In previous studies, using the cryosection immunogold technique, we have found that the M. tuberculosis phagosome exhibits delayed clearance of major histocompatability complex class I molecules and relatively weak staining for lysosomal membrane glycoproteins CD63, LAMP-1,
      The abbreviations used are:
      LAMP
      lysosome-associated membrane glycoprotein
      Alix
      programmed cell death 6-interacting protein
      Arf
      ADP-ribosylation factor
      Arl
      ADP-ribosylation factor-like
      BASP1
      brain acid-soluble protein 1
      BCG
      bacille Calmette-Guérin
      BiP
      Binding Protein
      CAP1
      adenylyl cyclase-associated protein 1
      CHMP4b
      charged multivesicular body protein 4b
      ER
      endoplasmic reticulum
      GFP
      green fluorescent protein
      GNAI2
      guanine nucleotide-binding protein Gi, α-2 subunit
      HB
      homogenization buffer
      HRP
      horseradish peroxidase
      IPTG
      isopropyl β-d-1-thiogalactopyranoside
      LAM
      lipoarabinomannan
      M6PR
      mannose 6-phosphate receptor
      Mtb-iGFP
      M. tuberculosis with inducible GFP expression
      MVB
      multivesicular body
      NPC1
      Niemann-Pick protein C1
      PMA
      phorbol 12-myristate 13-acetate
      PNS
      postnuclear supernate
      RPMI
      Roswell Park Memorial Institute
      SPFH
      stomatin, prohibitin, flotillin, HflK/C
      ATPase
      vacuolar ATPase
      VAT-1
      vesicle amine transport membrane protein-1
      VDAC
      voltage-dependent anion channel
      HI-FBS
      heat-inactivated fetal bovine serum
      GDI
      guanine dissociation inhibitor.
      1The abbreviations used are:LAMP
      lysosome-associated membrane glycoprotein
      Alix
      programmed cell death 6-interacting protein
      Arf
      ADP-ribosylation factor
      Arl
      ADP-ribosylation factor-like
      BASP1
      brain acid-soluble protein 1
      BCG
      bacille Calmette-Guérin
      BiP
      Binding Protein
      CAP1
      adenylyl cyclase-associated protein 1
      CHMP4b
      charged multivesicular body protein 4b
      ER
      endoplasmic reticulum
      GFP
      green fluorescent protein
      GNAI2
      guanine nucleotide-binding protein Gi, α-2 subunit
      HB
      homogenization buffer
      HRP
      horseradish peroxidase
      IPTG
      isopropyl β-d-1-thiogalactopyranoside
      LAM
      lipoarabinomannan
      M6PR
      mannose 6-phosphate receptor
      Mtb-iGFP
      M. tuberculosis with inducible GFP expression
      MVB
      multivesicular body
      NPC1
      Niemann-Pick protein C1
      PMA
      phorbol 12-myristate 13-acetate
      PNS
      postnuclear supernate
      RPMI
      Roswell Park Memorial Institute
      SPFH
      stomatin, prohibitin, flotillin, HflK/C
      ATPase
      vacuolar ATPase
      VAT-1
      vesicle amine transport membrane protein-1
      VDAC
      voltage-dependent anion channel
      HI-FBS
      heat-inactivated fetal bovine serum
      GDI
      guanine dissociation inhibitor.
      and LAMP-2 and the lysosomal acid protease cathepsin D (
      • Clemens D.L.
      Characterization of the Mycobacterium tuberculosis phagosome.
      ,
      • Clemens D.L.
      • Horwitz M.A.
      Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited.
      ,
      • Clemens D.L.
      • Horwitz M.A.
      The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin.
      ,
      • Clemens D.L.
      • Lee B.Y.
      • Horwitz M.A.
      Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7.
      ,
      • Clemens D.L.
      • Lee B.Y.
      • Horwitz M.A.
      Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate.
      ). Studies by other investigators have also demonstrated that M. tuberculosis and other mycobacterial species, including Mycobacterium bovis BCG, reside in phagosomes that resist acidification, are less mature, and less fusogenic with lysosomes than phagosomes containing inert particles (
      • Via L.E.
      • Deretic D.
      • Ulmer R.J.
      • Hibler N.S.
      • Huber L.A.
      • Deretic V.
      Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7.
      ,
      • Stewart G.R.
      • Patel J.
      • Robertson B.D.
      • Rae A.
      • Young D.B.
      Mycobacterial mutants with defective control of phagosomal acidification.
      ,
      • Sturgill-Koszycki S.
      • Schlesinger P.H.
      • Chakraborty P.
      • Haddix P.L.
      • Collins H.L.
      • Fok A.K.
      • Allen R.D.
      • Gluck S.L.
      • Heuser J.
      • Russell D.G.
      Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.
      ). These results are consistent with the hypothesis that M. tuberculosis and M. bovis BCG retard the maturation of their phagosomes along the endolysosomal pathway and reside in a compartment that has not matured fully to a phagolysosome (
      • Clemens D.L.
      • Horwitz M.A.
      Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited.
      ). Although the phagosomes of latex beads have been subjected to detailed proteomics analysis by Desjardins and co-workers (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ), a detailed proteomics study of the M. bovis BCG phagosome has not been reported previously.
      We describe in this study a novel method for the purification of the BCG phagosome from infected human macrophages, a detailed proteomics analysis of the BCG phagosome, and a comparison of the phagosome with latex bead phagosomes isolated from human macrophages. This study is the first comprehensive proteomics study of the M. bovis BCG phagosome and the first mass spectrometry-based proteomics study of the latex bead phagosome in human macrophages. We showed by Western immunoblotting that, relative to latex bead phagosomes, the BCG phagosome is relatively depleted in LAMP-2, NPC1, flotillin-1, vATPase, and syntaxin 3. Remarkably, by mass spectrometry, we documented a high degree of overlap in the set of proteins on BCG and latex bead phagosomes but also noteworthy differences. Novel proteins detected on the BCG phagosome but not on the latex bead phagosome include CD44, intercellular adhesion molecule 1, protein FAM3C, Ral-A/Ral-B, stress-induced phosphoprotein 1, band 4.1-like protein 3, septin-7, Ras GTPase-activating protein-like protein IQGAP1, Rab-6A, erlin-2, and tumor protein D54. Conversely, proteins identified on latex bead phagosomes but not on the BCG phagosome are β-galactosidase and sialate O-acetylesterase.

      RESULTS

      Purification of BCG Phagosomes and Latex Bead Phagosomes

      We initially attempted to purify BCG phagosomes using density gradient techniques described previously for bacterial phagosomes (
      • Via L.E.
      • Deretic D.
      • Ulmer R.J.
      • Hibler N.S.
      • Huber L.A.
      • Deretic V.
      Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7.
      ,
      • Lührmann A.
      • Haas A.
      A method to purify bacteria-containing phagosomes from infected macrophages.
      ). However, we observed that the buoyant density of BCG and of M. tuberculosis depends on the culture conditions and that bacteria initially grown on agar or in liquid medium become lighter after growth in macrophages. Whereas M. tuberculosis and BCG grown on 7H11 agar plates or in detergent-free 7H9 broth sedimented into 55% (w/v) sucrose, M. tuberculosis and BCG grown for 1 or more days within macrophages became much lighter and sedimented in the range of 31–35% sucrose. Therefore, purification strategies that rely on sedimentation of bacterial phagosomes through 55% sucrose as described previously for intracellular parasite phagosomes (
      • Via L.E.
      • Deretic D.
      • Ulmer R.J.
      • Hibler N.S.
      • Huber L.A.
      • Deretic V.
      Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7.
      ,
      • Lührmann A.
      • Haas A.
      A method to purify bacteria-containing phagosomes from infected macrophages.
      ) were not applicable to BCG or M. tuberculosis obtained from human macrophage culture. The buoyant density of BCG, assessed in iso-osmotic iodixanol density gradients, was also dramatically decreased after growth in macrophages. Whereas BCG freshly scraped from 7H11 agar plates exhibited a density of 1.114 on iodixanol gradients (corresponding to 20% iodixanol; Fig. 1, “sham” BCG phagosome gradients), BCG harvested after 3 days of growth in human THP-1 macrophages exhibited a much lighter density of 1.091 (corresponding to 12% iodixanol; Fig. 1, “live BCG-GFP”). Similarly, M. tuberculosis freshly harvested from agar plates exhibited a density of 1.126 in iodixanol gradients, whereas bacteria harvested after 1 day of growth in macrophage monolayers exhibited a density of 1.106 on iodixanol gradients. Killed BCG phagosomes exhibited a density similar to that of bacteria freshly harvested from plates (Fig. 1). This phenomenon is not attributable to selective uptake of less dense bacteria by macrophages because, in a separate experiment in which THP-1 macrophage monolayers were infected with agar plate-grown BCG that had been pelleted through 19% iodixanol (to remove less dense bacteria), the BCG harvested from the macrophages after 3 days of growth exhibited a density of 1.094 (corresponding to 13% iodixanol).
      Figure thumbnail gr1
      Fig. 1Sedimentation of M. bovis BCG phagosomes from THP-1 cells on iodixanol density gradients 3 days postinfection. A, human THP-1 macrophages were allowed to phagocytose live or killed M. bovis BCG-GFP or left untreated (SHAM), and a phagosomal fraction was purified 3 days postinfection. The sham phagosome preparations were processed identically to the other phagosome preparations except that M. bovis BCG was added to the macrophages at the end rather than the start of the incubation period and immediately prior to homogenization. Macrophages from all preparations were homogenized, PNS fractions were obtained by low speed centrifugation, and a phagosomal fraction was obtained by centrifugation through 15% sucrose onto 30% iodixanol. The BCG phagosomes (and free BCG in the case of the sham preparation) were collected from the interface and applied to linear iodixanol gradients. Gradient fractions were collected from the bottom, and aliquots representing equal fractions of the iodixanol gradient or the PNS (0.06% of the entire gradient or 0.06% of the total PNS, respectively) were analyzed by one-dimensional SDS-PAGE and Western immunoblotting. The relative number of BCG-GFP per gradient fraction was determined by mixing equal aliquots of each fraction with formaldehyde and counting the number of green fluorescent bacteria per 400× field by fluorescence microscopy. The BCG phagosomes are lighter than the majority of the mitochondria and ER and go to higher numbered fractions. Whereas LAMP-2 co-sediments with the BCG phagosomes, the mitochondrial antigen markers and the ER markers (calreticulin and BiP) are predominantly found in denser fractions. Whereas the mitochondrial marker VDAC and the ER marker BiP are abundant in the PNS, the majority of these organelles were removed in the purification step prior to the linear iodixanol gradient (low speed sedimentation through 15% sucrose) and thus are present at low levels in the iodixanol gradient fractions. S, sham; K, killed BCG; L, live BCG; M, molecular mass marker lane. B shows an independent replication of the purification of live and sham BCG phagosomes on an iodixanol gradient with analysis of gradient fractions by SDS-PAGE and Western immunoblotting for organelle markers. The BCG phagosomes co-elute with strong bands of LAMP-2 and cathepsin D. As is the case in A, the majority of ER and mitochondria are removed at the step prior to the linear iodixanol gradient. Some residual ER is apparent by calreticulin staining, mostly in fractions that are denser (lower numbered fractions) than the BCG phagosomes, although some calreticulin does co-elute. The mitochondria (stained by the mitochondrial antigens manganese superoxide dismutase (Mn-SOD) and VDAC) also elute in fractions that are denser than the BCG phagosomes. Clathrin (present on plasma membrane) and moesin (a cytoskeletal marker) are abundant in the PNS but are not detected by immunostaining in the peak fractions containing the BCG phagosomes.
      Because BCG became lighter with growth in macrophages, we purified BCG phagosomes from mouse J774 macrophages and from human THP-1 macrophages by using a combination of differential centrifugation and flotation on iodixanol density gradients. After obtaining a PNS, we removed the majority of contaminating organelles by low speed centrifugation (1000 × g for 45 min) through 15% sucrose onto a cushion of 30% iodixanol. Whereas mycobacterial phagosomes sediment through the 15% sucrose under these conditions, the majority of macrophage organelles and cytosolic constituents do not (
      • Sturgill-Koszycki S.
      • Schlesinger P.H.
      • Chakraborty P.
      • Haddix P.L.
      • Collins H.L.
      • Fok A.K.
      • Allen R.D.
      • Gluck S.L.
      • Heuser J.
      • Russell D.G.
      Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.
      ,
      • Chakraborty P.
      • Sturgill-Koszycki S.
      • Russell D.G.
      Isolation and characterization of pathogen-containing phagosomes.
      ). A substantial portion of the remaining contaminating organelles are removed by flotation of the phagosomes on an iodixanol density gradient, a technique that exploits the relatively unique low density of the mycobacteria in this iso-osmotic density gradient medium. Additional purification is obtained by sucrose density gradient sedimentation and the final low speed sedimentation, which pellets the bacterial phagosomes. In comparisons of true BCG phagosome and sham phagosome preparations run simultaneously under the same conditions, we observed relatively little contaminating protein in the sham phagosome preparations prepared from the density gradient fractions corresponding to those of the true BCG phagosome preparations (prepared from infected macrophages) (Fig. 2). We found that this technique also works well with mouse J774 macrophages, yielding a BCG phagosome preparation with more than 10-fold the protein content of that found in the corresponding sham phagosome preparation (supplemental Fig. 1).
      Figure thumbnail gr2
      Fig. 2Analysis of BCG phagosomes purified 3 days postinfection from THP-1 cells by SDS-PAGE and electron microscopy. A, BCG phagosomes purified from THP-1 cells 3 days after infection (BCG Phagosome) and sham phagosomes (Sham) prepared from identically treated, non-infected THP-1 cells to which BCG bacteria were added at the end rather than the beginning of the incubation period and immediately prior to homogenization were applied to a one-dimensional SDS-PAGE gradient gel and stained for protein by SYPRO Ruby. Masses of standard molecular mass markers (lane labeled “M”) are indicated in kDa. B and C, transmission electron microscopy of the PNS starting material (pelleted by centrifugation at 10,000 × g for 10 min) (B) and the final purified BCG-GFP phagosomal pellet (C) demonstrates that the purified phagosomes (C) have relatively little contamination and are considerably enriched in BCG phagosomes compared with the PNS starting material (B). Size bars, 1 µm.
      Proteins present in the SDS-PAGE gels of purified BCG phagosomes are almost exclusively host macrophage (human) proteins. We found that M. tuberculosis and BCG proteins are not extracted under the conditions that we use to extract host phagosomal proteins (heating at 100 °C for 5 min in SDS-PAGE sample buffer), and thus mycobacterial proteins do not contribute significantly to the protein staining seen in our SDS-PAGE gels of phagosomal preparations. This is probably attributable to the detergent-resistant, thick, waxy cell wall present on the mycobacteria. Likewise, we did not identify any mycobacterial proteins in our mass spectrometry-based proteomics analysis, although mycobacterial LAM was abundant in the preparations, and mycobacteria appeared to constitute the majority of the biomass as seen by electron microscopy of the purified phagosomes (Fig. 2).
      To assess the purity of our BCG phagosome preparations and to explore the presence or absence of selected antigenic proteins, we analyzed gradient fractions by Western immunoblotting. We observed that whereas the late endosomal-lysosomal marker LAMP-2 (Fig. 1, A and B) and the acid hydrolase cathepsin D (Fig. 1B) correspond well to the distribution of BCG-GFP from infected macrophages on the iodixanol gradient, the mitochondrial markers, VDAC (Fig. 1, A and B), the 60-kDa mitochondrial antigen (Fig. 1A), and manganese superoxide dismutase (Fig. 1B), were found in lower (more dense) fractions and were not detected by Western immunoblotting in the fractions containing the BCG phagosomes. Although the peak of the ER marker calreticulin was found in relatively dense fractions (Fig.1A, fractions 12–16 of the live BCG-GFP gradient, and Fig. 1B, fractions 19–23 of the live BCG-GFP gradient), this marker consistently trailed into the lighter fractions containing the BCG-GFP phagosomes (Fig. 1A, peak fraction 23 of the live BCG-GFP gradient and Fig. 1B, peak fraction 27 of the live BCG-GFP gradient). We observed a similar trailing of calnexin into the 3-day BCG phagosome fractions (data not shown) and a similar profile for LAMP-2, mitochondrial VDAC, and calreticulin relative to BCG phagosomes on iodixanol gradients prepared from THP-1 macrophages at 3 h postinfection (supplemental Fig. 2). We also observed an association of calreticulin and other ER markers with latex bead phagosomes purified by sucrose density flotation (supplemental Fig. 2), consistent with prior reports regarding highly purified latex bead phagosomes from mouse J774 macrophages (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ,
      • Gagnon E.
      • Duclos S.
      • Rondeau C.
      • Chevet E.
      • Cameron P.H.
      • Steele-Mortimer O.
      • Paiement J.
      • Bergeron J.J.
      • Desjardins M.
      Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages.
      ). Whether the presence of calreticulin and calnexin in regions of the iodixanol gradient containing BCG phagosomes and latex bead phagosomes reflects a specific contribution of ER to the phagosome, as has been reported in the case of phagosomes containing latex beads, killed bacteria, zymosan, (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ,
      • Gagnon E.
      • Duclos S.
      • Rondeau C.
      • Chevet E.
      • Cameron P.H.
      • Steele-Mortimer O.
      • Paiement J.
      • Bergeron J.J.
      • Desjardins M.
      Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages.
      ,
      • Giodini A.
      • Rahner C.
      • Cresswell P.
      Receptor-mediated phagocytosis elicits cross-presentation in nonprofessional antigen-presenting cells.
      ,
      • Ackerman A.L.
      • Kyritsis C.
      • Tampé R.
      • Cresswell P.
      Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.
      ), Toxoplasma gondii (
      • Goldszmid R.S.
      • Coppens I.
      • Lev A.
      • Caspar P.
      • Mellman I.
      • Sher A.
      Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii-infected dendritic cells.
      ), and M. tuberculosis (
      • Grotzke J.E.
      • Harriff M.J.
      • Siler A.C.
      • Nolt D.
      • Delepine J.
      • Lewinsohn D.A.
      • Lewinsohn D.M.
      The Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle.
      ), or instead reflects contamination (
      • Touret N.
      • Paroutis P.
      • Terebiznik M.
      • Harrison R.E.
      • Trombetta S.
      • Pypaert M.
      • Chow A.
      • Jiang A.
      • Shaw J.
      • Yip C.
      • Moore H.P.
      • van der Wel N.
      • Houben D.
      • Peters P.J.
      • de Chastellier C.
      • Mellman I.
      • Grinstein S.
      Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.
      ) remains to be determined. Clathrin, present on the plasma membrane and Golgi apparatus (
      • Royle S.J.
      The cellular functions of clathrin.
      ), was detected in the PNS but was absent from the gradient fractions containing the BCG phagosomes (Fig. 3B). Similarly, the cytoskeletal component moesin was stained intensely in the PNS but was relatively depleted in the gradient fractions corresponding to the BCG phagosomes (Fig. 1B).
      Figure thumbnail gr3
      Fig. 3Analysis of 3-day purified BCG phagosomes from THP-1 cells by Western immunoblotting. A, comparison of levels of LAMP-2, calnexin, 60-kDa mitochondrial antigen, and mycobacterial LAM. Samples containing 5–40 µg of protein from the PNS or BCG phagosomes prepared from THP-1 cells 3 days postinfection were analyzed by Western immunoblotting. Whereas LAMP-2 and mycobacterial LAM are enriched on the BCG phagosomes relative to the amount present in the PNS per µg of protein, calnexin is present at a markedly reduced level, and the 60-kDa mitochondrial antigen (Mito) is not detected in the purified BCG phagosome fraction. B, comparison of levels of clathrin, calnexin, mitochondrial antigen (Mito), LAM, Rab-11, and galectin-1. A separate Western immunoblot experiment demonstrates the enrichment of LAM in the BCG phagosome preparation and the persistence of low levels of calnexin relative to the levels present in the PNS. In contrast, clathrin, the mitochondrial antigen, Rab-11, and galectin-1 are detected in the PNS but not in the BCG phagosome preparation.
      We also examined the purity of our BCG phagosome preparations at the ultrastructural level by transmission electron microscopy (Fig. 2, B and C) and observed that the BCG phagosomes purified from THP-1 cells exhibit a high level of purity with relatively little extraneous material (Fig. 2C) and a high level of enrichment over the PNS starting material (Fig. 2B). In addition, we performed a quantitative analysis of the phagosomal purity of our 3-day BCG phagosomes by using the radioisotopic mixing method described by Chakraborty et al. (
      • Chakraborty P.
      • Sturgill-Koszycki S.
      • Russell D.G.
      Isolation and characterization of pathogen-containing phagosomes.
      ) and obtained calculated purities of 82.5 and 92%, respectively.
      To analyze the degree to which selected proteins are associated with or excluded from the BCG and latex bead phagosomes, we loaded SDS-PAGE gels with known amounts of protein from either the PNS or purified phagosomal preparations and analyzed the proteins present in the sample by Western immunoblotting. The purified BCG phagosomes exhibited an enrichment in mycobacterial LAM, a strong presence of LAMP-2, the absence of the mitochondrial 60-kDa marker, and a relative depletion in calnexin relative to the levels found in the PNS (Fig. 3A). Plasma membrane-associated proteins galectin-1 and clathrin were abundant in the PNS but not detected by Western immunoblotting in the BCG phagosome preparation (Fig. 3B). Rab-11, a marker of recycling endosomes, was detected in the PNS but was relatively depleted in the BCG phagosome preparation (Fig. 3B). We also compared the relative enrichment or depletion of selected markers on BCG phagosomes and latex bead phagosomes isolated from THP-1 macrophages at 3 days postinfection by analyzing equivalent amounts of phagosomal proteins by Western immunoblotting (Fig. 4). Whereas NPC1 and LAMP-2 were greatly enriched on the latex bead phagosomes, their relative abundance on BCG phagosomes was similar to that in the PNS. Whereas flotillin-1 and vATPase 6E were greatly enriched on latex bead phagosomes (relative to the PNS), they were markedly depleted on BCG phagosomes (Fig. 4). The membrane trafficking protein syntaxin 3 also was depleted on BCG phagosomes relative to its abundance on latex bead phagosomes and in the PNS (Fig. 4). The cytoskeletal protein stathmin was present in the PNS but was undetectable by Western immunoblotting in both the BCG and latex bead phagosome preparations (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Analysis of 3-day purified BCG phagosomes, latex bead phagosomes, and PNS by Western immunoblotting. Samples containing 5–40 µg of protein from the PNS, purified BCG phagosomes, or purified latex bead phagosomes prepared from THP-1 cells 3 days postinfection were analyzed by Western immunoblotting. NPC1, LAMP-2, and syntaxin 3 are enriched on the latex bead phagosomes relative to the levels observed on BCG phagosomes and the PNS. Flotillin-1 and the vATPase are greatly enriched on the latex bead phagosome and relatively scarce on the BCG phagosome. The cytoskeletal protein stathmin, on the other hand, is detected in the PNS but not in the latex bead or BCG phagosome preparations.

      Proteomics Analysis of BCG Phagosomes

      Proteins present in BCG phagosomes and latex bead phagosomes purified from THP-1 cells infected for 3 h, 1 day, 3 days, or 5 days were separated by SDS-PAGE on gradient gels, the gel slices were digested with trypsin, and the resulting peptides were extracted and analyzed by nano-HPLC-MS/MS. The constituent proteins were identified by comparison against the human genome. Because of the complexity of these mixtures even after one-dimensional gel separations and to obtain an in-depth coverage of the proteins in these preparations, we subjected the trypsin-digested gel slices to HPLC-MS/MS analysis. A total of 475 different human proteins were identified in at least one of the BCG or latex bead phagosome preparations by HPLC-MS/MS analysis (supplemental Table 1). In addition, we identified 14 mouse proteins on one-dimensional and two-dimensional gels using the same methods in a limited analysis of BCG phagosomes isolated 1 day postinfection of J774 macrophages (supplemental Fig. 1 and Tables 2 and 3). A total of 447 human proteins were identified in one or more of the five BCG phagosome preparations, and a total of 289 were identified in one or more of the three latex bead phagosome preparations; 260 proteins were also identified one or more times in both BCG and latex bead phagosome preparations. To assess the consistency with which individual proteins were identified in the BCG phagosome preparations, we ranked each protein based on how often it was detected in the five BCG preparations (i.e. four time points with one independent biological replicate of the 3-day time point; supplemental Table 1). We identified 32 host proteins at all four time points in all five of the BCG phagosome preparations (supplemental Table 1). All 32 of these were also identified in at least one latex bead phagosome preparation, and 16 were identified in all three of the latex bead phagosome preparations. We identified 98 proteins in at least four BCG phagosome preparations of which all but 13 were found in at least one latex bead phagosome preparation (Table I, first 13 proteins), and we identified 178 proteins in at least three of the five BCG phagosome preparations of which all but 32 were found in at least one of the latex bead phagosome preparations (Table I, all proteins). These data indicate a high level of concordance for the proteins present in the BCG and latex bead phagosome preparations. Because only the 3-day time point was assessed more than once, we did not attempt to evaluate changes in phagosome composition over time; instead we used the data to identify a set of proteins that were consistently detected on the BCG phagosome across multiple preparations.
      Table IProteins identified in the majority of BCG phagosome preparations that were not identified in any latex bead phagosome preparations
      Protein identifiedSwiss-Prot accession no.Reported location
      Except where indicated by referenced citation, “Reported location” and “Physiological function” are abstracted from the Swiss-Prot database.
      Physiological function
      Except where indicated by referenced citation, “Reported location” and “Physiological function” are abstracted from the Swiss-Prot database.
      No. BCG+Spectral/peptide counts
      Spectral counts refer to all acquired precursor ions for a particular protein, whereas peptide counts only list the number of unique peptide sequences observed per protein (for correlation of spectral counts to the relative protein concentration see Liu et al. (83)). For BCG, these are listed for 3 h, 1 days, 3 days (a), 3 days (b), and 5 days, respectively. 3 days (a) and 3 days (b) are biological replicates.
      3 h1 day3 days (a)3 days (b)5 days
      CD44 antigenP16070PMHA receptor401/11/11/12/2
      Erlin-2 (SPFH domain-containing protein 2)O94905ER membrane, PMLipid raft-associated43/305/11/13/2
      Golgi apparatus protein 1 (E-selectin ligand 1)Q92896Golgi and PMBinds fibroblast growth factor and E-selectin402/24/424/2411/11
      Intercellular adhesion molecule 1P05362PMLigand for LFA-1 protein401/12/23/31/1
      Protein FAM3CQ92520Putative macrophage-secreted proteinUnknown; member of cytokine-like gene family; may be involved in cell differentiation and proliferation during embryogenesis404/42/24/44/4
      Ras GTPase-activating-like protein IQGAP1P46940MembraneIntegrates Ca2+/calmodulin and Cdc42 signaling403/35/49/914/14
      Ras-related protein Rab-6AP20340GolgiGolgi to ER traffic402/22/27/76/6
      Stress-induced-phosphoprotein 1P31948Cytoplasm, nucleusMediates the association of chaperones Hsc70 and Hsc90406/62/25/58/8
      Transmembrane emp24 domain-containing protein 9Q9BVK6ER membraneUnknown401/11/12/21/1
      Tubulin α-1C chainQ9BQE3CytoplasmCytoskeleton407/73/37/72/2
      Tubulin β chainP07437CytoplasmCytoskeleton4013/1212/1112/124/4
      Tumor protein D54O43399UnknownUnknown401/12/23/31/1
      Very long-chain-specific acyl-CoA dehydrogenase, mitochondrialP49748MitochondrionFatty acid β-oxidation system404/44/41/12/2
      ADP/ATP translocase 2P05141MitochondrionATP/ADP translocation34/43/31/100
      α-Mannosidase 2Q16706GolgiN-Glycosylation302/206/612/12
      Band 4.1-like protein 3Q9Y2J2CytoplasmLinks membrane to cytoskeleton3001/11/12/2
      Charged multivesicular body protein 4bQ9H444Cytoplasm, endosomeMVB formation and sorting of endosomal cargo proteins into MVBs3005/32/21/1
      Glutamate dehydrogenase 1, mitochondrialP00367MitochondrionOxidative deamination of glutamate to α-ketoglutarate34/32/22/200
      Guanine nucleotide-binding protein Gi/Gs/Gt subunit β-1P62873MembraneTransmembrane signaling33/3002/22/2
      Integrin α5P08648PMReceptor for fibronectin and fibrinogen, present in endocytic pathway (
      • Shakibaei M.
      • Zimmermann B.
      • Scheller M.
      Endocytosis of integrin alpha 5 beta 1 (fibronectin receptor) of mouse peritoneal macrophages in vitro: an immunoelectron microscopic study.
      )
      31/13/31/100
      Nucleobindin-1Q02818Golgi, cytoplasm, membraneCalcium homeostasis303/306/68/8
      Platelet endothelial cell adhesion moleculeP16284PMCell adhesion; signal transduction3005/54/45/5
      Ras-related protein Rab-14P61106MembraneVesicular trafficking301/11/12/20
      Septin-7Q16181CytoplasmGTPase; associates with actin stress fibers; involved in cytokinesis and exocytosis3002/22/24/4
      Synaptic vesicle membrane protein VAT-1 homologQ99536MembraneUnknown; ATPase with homology to oxidoreductase and ξ-crystallin32/2001/12/2
      Vacuolar proton pump subunit C 1P21283Intracellular compartments, endosome-lysosomeCompartment acidification36/6001/11/1
      Vesicle-trafficking protein Sec22bO75396ER-Golgi-intermediate compartment, melanosomeER to Golgi targeting and fusion3002/22/23/3
      60 S ribosomal protein L35P42766
      Proteins identified by single peptide assignments.
      CytoplasmProtein synthesis301/101/11/1
      60 S ribosomal protein L8P62917
      Proteins identified by single peptide assignments.
      CytoplasmProtein synthesis3001/11/11/1
      Macrophage migration-inhibitory factorP14174
      Proteins identified by single peptide assignments.
      Macrophage-secreted proteinProinflammatory immune modulator301/11/11/10
      Ras-related protein Ral-B
      Ral-B (P11234) could not be differentiated from Ral-A (also see supplemental Table 1); 78% sequence identity between Ral-B and Ral-A (P11233).
      P11234
      Proteins identified by single peptide assignments.
      MembraneSignal transduction; regulation of exocytosis; activation of IκB kinase TBK131/1001/11/1
      Translocon-associated protein subunit αP43307
      Proteins identified by single peptide assignments.
      ERBinds Ca2+ to ER membrane; regulates retention of ER-resident proteins31/101/101/1
      a Except where indicated by referenced citation, “Reported location” and “Physiological function” are abstracted from the Swiss-Prot database.
      b Spectral counts refer to all acquired precursor ions for a particular protein, whereas peptide counts only list the number of unique peptide sequences observed per protein (for correlation of spectral counts to the relative protein concentration see Liu et al. (
      • Liu H.
      • Sadygov R.G.
      • Yates 3rd, J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      )). For BCG, these are listed for 3 h, 1 days, 3 days (a), 3 days (b), and 5 days, respectively. 3 days (a) and 3 days (b) are biological replicates.
      c Proteins identified by single peptide assignments.
      d Ral-B (P11234) could not be differentiated from Ral-A (also see supplemental Table 1); 78% sequence identity between Ral-B and Ral-A (P11233).
      Although our proteomics analysis of BCG phagosomes and latex bead phagosomes indicate that there are more proteins in common than not for these two compartments, the distinctions between the two compartments may reflect important differences in cell biology. Included among the 32 proteins present in BCG phagosomes and undetected in latex bead phagosomes (Table I) were (a) proteins involved in membrane trafficking, vesicle-trafficking protein Sec22b, charged multivesicular body protein 4b (CHMP4b), Ras-related proteins Rab-14, Rab-6A, and Ral-A/Ral-B; (b) membrane proteins CD44 and intercellular adhesion molecule 1; (c) proteins thought to be involved in signal transduction or regulation of the cytoskeleton, Ras GTPase-activating-like protein IQGAP1, stress-induced phosphoprotein 1, septin-7, and band 4.1-like protein 3; and (d) the macrophage-secreted protein, macrophage migration-inhibitory factor, and the putative secreted protein FAM3C (Table I). Attempts to validate many of the proteins listed in Table I by Western immunoblotting were unsuccessful because of problems with a lack of sensitivity and specificity of the available antibodies (i.e. the available antibodies were either non-reactive with the proteins on blots, or they had excessive cross-reactivity with mycobacterial antigens). However, we confirmed the presence of IQGAP1 on M. bovis BCG phagosomes but not latex bead phagosomes by Western immunoblotting (supplemental Fig. 3), and we confirmed by immunofluorescence microscopy the differential presence of FAM3C on M. bovis BCG phagosomes but not latex bead phagosomes (supplemental Fig. 4).
      Of the 289 proteins identified in at least one of the latex bead phagosome preparations, 34 were identified in all three latex bead phagosome preparations. All 34 of these were detected in at least one BCG phagosome preparation, and 29 were identified in at least four of the five BCG phagosome preparations (supplemental Table 1), demonstrating again the high concordance of proteins on the latex bead and BCG phagosome preparations. Of 104 host proteins found in at least two of the three latex bead phagosome preparations, only two were not found in any of the BCG phagosome preparations: the lysosomal enzymes β-galactosidase and sialate O-acetyltransferase. We confirmed by immunofluorescence microscopy that β-galactosidase was present on latex bead phagosomes but not on BCG phagosomes at 3-days postinfection in THP-1 cells (supplemental Fig. 4). In addition, the lysosomal hydrolase glucosylceramidase was found in two of the latex bead phagosome preparations (3 h and 5 day) but only in the 3-h BCG phagosome preparation. The lower representation or absence of the lysosomal enzymes glucosylceramidase, β-galactosidase, and sialate O-acetyltransferase may reflect a lesser degree of fusion of the BCG phagosome with lysosomes relative to the latex bead phagosome. Of the 104 host proteins identified in at least two of the three latex bead phagosome preparations, 61 were also identified in at least four of the five BCG phagosome preparations (Table II).
      Table IIProteins shared by BCG and latex bead phagosomes (proteins identified in at least four of five BCG and at least two of three latex bead phagosome preparations)
      Protein identifiedSwiss-Prot accession no.No. BCG
      No. BCG indicates the number of BCG phagosome (out of five total) in which the indicated protein was identified.
      No. bead
      No. bead indicates the number of latex bead phagosome preparations (out of three total) in which the indicated protein was identified.
      Spectral/peptide count
      Spectral counts refer to all acquired precursor ions for a particular protein, whereas peptide counts only list the number of unique peptide sequences observed per protein in the phagosome preparation (for correlation of spectral counts to the relative protein concentration see Liu et al. (83)). For BCG, these are listed for 3 h, 1 days, 3 days (a), 3 days (b), and 5 days, respectively. For latex beads, these are listed for 3 h, 3 days, and 5 days, respectively. The spectral counts for the 5 BCG phagosome preparations are listed together and are separated by a slash (/) from the peptide counts of the same 5 preparations, which are listed in the same order. The same is done for the spectral and peptide counts, respectively, for the 3 latex bead phagosome preparations.
      Reported location
      Reported location and physiological function are abstracted from the Swiss-Prot on-line database except where indicated by cited references.
      Physiological function
      Reported location and physiological function are abstracted from the Swiss-Prot on-line database except where indicated by cited references.
      BCGBead
      60-kDa heat shock protein, mitochondrialP10809539,19, 15, 2, 18/9, 19, 14, 2,1812, 16, 5/12, 16, 5Mitochondrion, secretory granulesChaperone; extramitochondrial localization reported (
      • Brudzynski K.
      • Martinez V.
      • Gupta R.S.
      Secretory granule autoantigen in insulin-dependent diabetes mellitus is related to 62 kDa heat-shock protein (hsp60).
      ,
      • Cechetto J.D.
      • Soltys B.J.
      • Gupta R.S.
      Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.
      ,
      • Brudzynski K.
      • Martinez V.
      • Gupta R.S.
      Immunocytochemical localization of heat-shock protein 60-related protein in beta-cell secretory granules and its altered distribution in non-obese diabetic mice.
      )
      78-kDa glucose-regulated proteinP11021535, 16, 6, 8, 13/5, 16, 6, 8, 135, 8, 16/5, 8, 16ER, melanosomeChaperone; member of heat shock protein 70 family
      ATP synthase subunit αP257055313, 12, 2, 4, 6/13, 12, 2, 4, 69, 7, 2/9, 7, 2Mitochondrion inner membraneIon transport; generation of ATP
      Aminopeptidase NP151445313, 8, 31, 20, 21/13, 8, 20, 20, 219, 17, 9/9, 12, 9PMProtein degradation
      CalnexinP27824531, 2, 2, 1, 2/1, 2, 2, 1, 21, 1, 3/1, 1, 3ERChaperone; protein synthesis and folding
      EndoplasminP14625534, 11, 10, 14, 13/4, 11, 10, 14, 139, 10, 14/8, 9, 13ERProtein synthesis and folding
      Integrin β2P05107535, 7, 8, 6, 12/5, 7, 8, 6, 123, 2, 2/3, 2, 2PMCell surface adhesion glycoprotein
      Lysosome membrane protein 2Q141085315, 3, 9, 12, 3/13, 3, 9, 12, 312, 5, 2/11, 5, 2LysosomeLysosomal receptor; scavenger receptor class B member 2
      Lysosome-associated membraneglycoprotein 1P11279534, 2, 4, 6, 3/4, 2, 3, 6, 34, 3, 2/4, 3, 2Lysosome, endosome, PMPresents carbohydrate ligands to selectins
      Lysosome-associated membrane glycoprotein 2P13473533, 1, 4, 2, 2/3, 1, 3, 2, 23, 5, 1/2, 3, 1Lysosome, endosome, PMProtects lysosomal membrane from autodigestion
      MoesinP26038533, 8, 3, 9, 12/3, 8, 3, 9, 124, 7, 14/4, 7, 14CytoplasmConnections of membrane to cytoskeleton
      Peroxiredoxin-1Q06830531, 2, 2, 6, 8/1, 2, 2, 5, 72, 1, 11/2, 1, 10Cytoplasm, melanosomeRedox regulation; detoxification
      Protein-disulfide isomeraseP07237533, 2, 5, 9, 7/3, 2, 5, 9, 72, 7, 11/2, 7, 11ERDisulfide bond rearrangement
      Protein-disulfide isomerase A3P30101531, 2, 10, 9, 10/1, 2, 10, 9, 102, 7, 7/2, 6, 7ERDisulfide bond rearrangement
      Putative elongation factor 1α-like 3Q5VTE0532, 5, 4, 3, 5/2, 5, 4, 3, 54, 8, 7/4, 7, 5CytoplasmProtein biosynthesis (by similarity)
      Ras-related protein Rab-5CP51148534, 4, 5, 8, 3/4, 4, 5, 7, 34, 6, 1/4, 5, 1Early endosomeVesicular trafficking
      Acid ceramidaseQ13510522, 1, 2, 2, 2/2, 1, 2, 2, 22, 2, 0/2, 2, 0LysosomeHydrolyzes ceramide into sphingosine and free fatty acid
      CD63 antigenP08962522, 4, 2, 3, 2/2, 4, 2, 3, 23, 4, 0/3, 3, 0Late endosome-lysosomeUnknown; may regulate transport of other proteins
      Coronin-1AP31146522, 1, 3, 3, 2/2, 1, 3, 3, 20, 4, 4/0, 3, 4CytoplasmCytoskeleton component; important in membrane invaginations, protrusions, and cell locomotion
      Erythrocyte band 7 integral membrane protein (stomatin)P27105521, 2, 2, 3, 4/1, 2, 2, 3, 46, 6, 0/6, 6, 0PM, melanosomeRegulates cation conductance; associates with actin and lipid rafts
      Flotillin-1O75955523, 1, 7, 1, 1/3, 1, 7, 1, 14, 9, 0/3, 9, 0PM, phagosomal membraneLipid rafts; clathrin-independent endocytosis; formation of caveolae or caveolae-like vesicles
      Fructose-bisphosphate aldolase AP04075523, 8, 5, 14, 15/3, 8, 5, 12, 154, 0, 14/4, 0, 13CytoplasmMetabolism; interacts with cytoskeleton (
      • Schindler R.
      • Weichselsdorfer E.
      • Wagner O.
      • Bereiter-Hahn J.
      Aldolase-localization in cultured cells: cell-type and substrate-specific regulation of cytoskeletal associations.
      )
      Heat shock protein HSP90-βP08238525, 10, 16, 22, 10/5, 10, 14, 22, 103, 0, 18/3, 0, 17Cytoplasm, melanosomeChaperone
      Proactivator polypeptide (prosaposin)P07602524, 1, 2, 4, 1/4, 1, 2, 4, 13, 3, 0/3, 3, 0LysosomeSphingolipid degradation
      Ras-related protein Rab-11BQ15907521, 2, 2, 1, 1/1, 2, 2, 1, 11, 1, 0/1, 1, 0Recycling endosome, PMRegulation of endosomal recycling
      Stress-70 protein, mitochondrialP386465212, 3, 8, 5, 4/12, 3, 8, 5, 42, 6, 0/2, 6, 0Mitochondrion, cell surfaceChaperone; control of cell proliferation and cellular aging
      Thioredoxin-dependent peroxide reductase, mitochondrialP30048522, 1, 1, 1, 1/1, 1, 1, 1, 11, 1, 0/1, 1, 0Mitochondrion, early endosomeRedox regulation; presence in endosomes reported (
      • Liu L.
      • Yang C.
      • Yuan J.
      • Chen X.
      • Xu J.
      • Wei Y.
      • Yang J.
      • Lin G.
      • Yu L.
      RPK118, a PX domain-containing protein, interacts with peroxiredoxin-3 through pseudo-kinase domains.
      )
      Vacuolar ATP synthase subunit BP212815210, 2, 3, 7, 2/10, 2, 3, 7, 28, 15, 0/8, 11, 0Endosome-lysosomeCompartment acidification
      Actin, cytoplasmic 1P60709435, 0, 16, 16, 49/5, 0, 12, 13, 163, 2, 18/3, 2, 13CytoplasmCytoskeleton
      Annexin A2P07355435, 0, 4, 9, 9/5, 0, 4, 9, 915, 8, 5/13, 6, 5Secreted, extracellular matrix, basement membraneMay cross-link PM phospholipids with actin cytoskeleton and be involved in exocytosis
      Brain acid-soluble protein 1P80723434, 0, 6, 3, 4/4, 0, 5, 3, 43, 5, 1/3, 5, 1MembraneSignaling; regulation of actin dynamics; present in lipid rafts; has calmodulin binding site; is substrate for PKC
      Cathepsin DP07339436, 0, 5, 8, 9/5, 0, 5, 8, 89, 15, 5/8, 11, 5LysosomeProtein degradation
      Glyceraldehyde-3-phosphate dehydrogenaseP04406432, 0, 3, 3, 7/2, 0, 3, 3, 73, 3, 5/3, 3, 5Cytoplasm, membrane-associatedGlycolysis; membrane fusion; microtubule bundling; phosphotransferase activity; nuclear RNA export; DNA replication and DNA repair (
      • Sirover M.A.
      New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.
      )
      Heat shock cognate 71-kDa proteinP11142436, 0, 9, 22, 8/6, 0, 9, 22, 83, 5, 11/3, 5, 11Cytoplasm, melanosomeChaperone
      Palmitoyl-protein thioesterase 1P50897433, 0, 3, 3, 4/3, 0, 3, 3, 44, 6, 1/4, 5, 1LysosomeLipoprotein degradation
      Plastin-2P13796430, 10, 2, 12, 14/0, 10, 2, 12, 142, 3, 11/2, 3, 11CytoplasmCytoskeleton
      Ras-related protein Rab-7AP51149432, 0, 5, 4, 7/2, 0, 5, 4, 73, 3, 2/3, 3, 2Late endosomeLate endosomal transport; phagosome maturation
      Vacuolar ATP synthase catalytic subunit AP38606433, 0, 1, 7, 4/3, 0, 1, 7, 44, 12, 1/4, 10, 1Endosome-lysosomeCompartment acidification
      VimentinP08670430, 5, 9, 2, 5/0, 5, 9, 2, 57, 8, 1/7, 8, 1CytoplasmCytoskeleton; intermediate filament
      Voltage-dependent anion-selective channel protein 1P21796434, 0, 2, 5, 7/4, 0, 1, 5, 72, 3, 5/2, 3, 5Mitochondrion, PMChannel for small hydrophilic molecules; present in secretory pathway and PM (
      • Buettner R.
      • Papoutsoglou G.
      • Scemes E.
      • Spray D.C.
      • Dermietzel R.
      Evidence for secretory pathway localization of a voltage-dependent anion channel isoform.
      )
      Voltage-dependent anion-selective channel protein 2P45880432, 0, 3, 2, 5/2, 0, 3, 2, 52, 1, 2/2, 1, 2MitochondrionChannel for small hydrophilic molecules
      10-kDa heat shock protein, mitochondrialP61604426, 6, 4, 4, 0/6, 5, 4, 4, 03, 5, 0/3, 5, 0Mitochondrion and non-mitochondrial sitesCo-chaperone for Hsp60 in the protein folding process; also identified in non-mitochondrial sites, including secretory granules (
      • Sadacharan S.K.
      • Cavanagh A.C.
      • Gupta R.S.
      Immunoelectron microscopy provides evidence for the presence of mitochondrial heat shock 10-kDa protein (chaperonin 10) in red blood cells and a variety of secretory granules.
      )
      40 S ribosomal protein SA (laminin receptor-1)P08865420, 1, 2, 3, 5/0, 1, 1, 3, 50, 1, 3/0, 1, 3CytoplasmProtein synthesis; cell adhesion
      ADP-ribosylation factor-like protein 8B
      Arl 8B (Q9NVJ2) could not be differentiated from ARL 8A (Q96BM9) (also see supplemental Table 1) in three of four BCG phagosome preparations and one of two latex bead phagosome preparations.
      Q9NVJ2423, 1, 1, 1, 0/3, 1, 1, 1, 02, 1, 0/2, 1, 0Late endosome-lysosomeEndosome and lysosome motility
      α-EnolaseP06733420, 1, 2, 6, 7/0, 1, 2, 6, 71, 0, 3/1, 0, 3Cytoplasm, PM, myofibril, sarcomereCarbohydrate degradation; glycolysis; promotes vacuole fusion in yeast; may also function as a plasminogen receptor and activator on the PM
      Cathepsin ZQ9UBR2423, 0, 1, 3, 2/3, 0, 1, 3, 22, 2, 0/2, 2, 0LysosomeCarboxydipeptidase
      Cofilin-1P23528421, 2, 0, 2, 1/1, 2, 0, 2, 11, 0, 2/1, 0, 2CytoplasmControls actin polymerization
      GelsolinP06396420, 1, 2, 8, 5/0, 1, 2, 8, 51, 0, 7/1, 0, 7CytoplasmCytoskeleton
      Hypoxia up-regulated protein 1Q9Y4L1420, 2, 1, 2, 7/0, 1, 1, 2, 71, 0, 3/1, 0, 3ER lumenChaperone; induced by hypoxia
      Lysosomal acid phosphataseP11117420, 1, 2, 1, 3/0, 1, 2, 1, 33, 5, 0/3, 5, 0LysosomePhosphate monoester hydrolysis
      Lysosomal α-glucosidaseP10253422, 0, 2, 1, 1/2, 0, 2, 1, 13, 2, 0/3, 2, 0LysosomeGlycogen degradation
      Malate dehydrogenase, mitochondrialP409264211, 0, 6, 2, 8/11, 0, 5, 2, 85, 5, 0/5, 5, 0MitochondrionGlycolysis; tricarboxylic acid cycle
      Myosin-9P35579420, 14, 5, 29, 15/0, 14, 5, 29, 150, 7, 34/0, 7, 34CytoplasmCytoskeleton
      N-Acetylglucosamine-6-sulfataseP15586428, 1, 1, 3, 0/8, 1, 1, 3, 03, 2, 0/3, 2, 0LysosomeHeparan sulfate/keratan sulfate hydrolysis
      NicastrinQ92542422, 1, 2, 2, 0/2, 1, 2, 2, 04, 3, 0/4, 3, 0Golgi, ER, PM, melanosomeComponent of the γ-secretase complex
      Protein-disulfide isomerase A6Q15084420, 3, 1, 4, 6/0, 3, 1, 4, 61, 0, 2/1, 0, 2ER, ER-Golgi intermediate compartment, melanosomeDisulfide bond rearrangement
      Ras-related protein Rab-21Q9UL25421, 0, 2, 2, 1/1, 0, 2, 2, 11, 2, 0/1, 2, 0EndosomesControls traffic of β1 integrins
      Sulfide:quinone oxidoreductase, mitochondrialQ9Y6N5422, 0, 6, 4, 6/2, 0, 6, 4, 62, 2, 0/2, 2, 0MitochondrionHydrogen sulfide oxidation
      Vacuolar proton pump subunit d 1P61421423, 0, 6, 5, 1/3, 0, 5, 5, 14, 5, 0/4, 4, 0Intracellular compartments, endosome-lysosomeCompartment acidification
      Citrate synthase, mitochondrialO75390
      Proteins identified by single peptide assignments.
      421, 0, 1, 1, 1/1, 0, 1, 1, 11, 1, 0/1, 1, 0MitochondrionCarbohydrate metabolism; tricarboxylic acid cycle
      Interferon-induced transmembrane protein 3
      Interferon-induced transmembrane protein 3 (Q01628) could not be differentiated from interferon-induced transmembrane protein 1 (P13164) or interferon-induced transmembrane protein 2 (Q01629) (also see supplemental Table 1).
      Q01628
      Proteins identified by single peptide assignments.
      421, 1, 1, 1, 0/1, 1, 1, 1, 01, 1, 0/1, 1, 0PM, Golgi, integral membrane proteinCell differentiation and homing; immune response
      a No. BCG indicates the number of BCG phagosome (out of five total) in which the indicated protein was identified.
      b No. bead indicates the number of latex bead phagosome preparations (out of three total) in which the indicated protein was identified.
      c Spectral counts refer to all acquired precursor ions for a particular protein, whereas peptide counts only list the number of unique peptide sequences observed per protein in the phagosome preparation (for correlation of spectral counts to the relative protein concentration see Liu et al. (
      • Liu H.
      • Sadygov R.G.
      • Yates 3rd, J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      )). For BCG, these are listed for 3 h, 1 days, 3 days (a), 3 days (b), and 5 days, respectively. For latex beads, these are listed for 3 h, 3 days, and 5 days, respectively. The spectral counts for the 5 BCG phagosome preparations are listed together and are separated by a slash (/) from the peptide counts of the same 5 preparations, which are listed in the same order. The same is done for the spectral and peptide counts, respectively, for the 3 latex bead phagosome preparations.
      d Reported location and physiological function are abstracted from the Swiss-Prot on-line database except where indicated by cited references.
      e Arl 8B (Q9NVJ2) could not be differentiated from ARL 8A (Q96BM9) (also see supplemental Table 1) in three of four BCG phagosome preparations and one of two latex bead phagosome preparations.
      f Proteins identified by single peptide assignments.
      g Interferon-induced transmembrane protein 3 (Q01628) could not be differentiated from interferon-induced transmembrane protein 1 (P13164) or interferon-induced transmembrane protein 2 (Q01629) (also see supplemental Table 1).
      Noteworthy proteins identified in the BCG phagosome included the following.

      Endosomal-Lysosomal Proteins

      Among the proteins found in at least four of the five BCG phagosome preparations and at least two of the three latex bead phagosome preparations were several well known endolysosomal proteins: the lysosomal membrane glycoproteins CD63, LAMP-1, LAMP-2, and lysosome membrane protein 2; subunit B of the vATPase; cathepsin D; cathepsin Z; palmitoyl-protein thioesterase; lysosomal acid phosphatase; N-acetylglucosamine-6-sulfatase; lysosomal α-glucosidase; acid ceramidase; and prosaposin. We detected the mannose 6-phosphate receptor-binding protein (which interacts with mannose 6-phosphate receptor (M6PR)) in four of the five BCG phagosome preparations and in only one of the latex bead phagosome preparations, and we detected the cation-independent M6PR in two of the five BCG preparations but in none of the latex bead phagosome preparations. Association of the M6PR and the M6PR-binding protein with the BCG phagosome may reflect the BCG phagosome being in a less mature, late endosomal-like compartment as opposed to a phagolysosomal compartment.

      Multivesicular Body (MVB) and Exosome Pathway

      In addition to many classical components of the endosomal-lysosomal pathway, we also identified many components of the more recently described MVB-exosome pathway in both BCG and latex bead phagosomes. For example, we identified programmed cell death 6-interacting protein (Alix) in both the 3- and 5-day BCG phagosomes (supplemental Table 1). This protein interacts with CHMP4b to promote the formation of multivesicular bodies (
      • Katoh K.
      • Shibata H.
      • Suzuki H.
      • Nara A.
      • Ishidoh K.
      • Kominami E.
      • Yoshimori T.
      • Maki M.
      The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.
      ,
      • Katoh K.
      • Shibata H.
      • Hatta K.
      • Maki M.
      CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms.
      ). We identified CHMP4b in all 3- and 5-day BCG phagosomes but not in any of the latex bead phagosomes (supplemental Table 1). Alix has also been identified on latex bead phagosomes purified from mouse macrophages (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ). To our knowledge, CHMP4b has not previously been identified in proteomics studies of phagosomes. MVBs are at the intersection of the endocytic pathway and the exosome pathway (
      • Février B.
      • Raposo G.
      Exosomes: endosomal-derived vesicles shipping extracellular messages.
      ). Thus, interaction of the phagosome with the MVB allows antigen processing and presentation via the exosome pathway. Many additional proteins that have been identified as major constituents of exosomes (
      • Février B.
      • Raposo G.
      Exosomes: endosomal-derived vesicles shipping extracellular messages.
      ,
      • Wubbolts R.
      • Leckie R.S.
      • Veenhuizen P.T.
      • Schwarzmann G.
      • Möbius W.
      • Hoernschemeyer J.
      • Slot J.W.
      • Geuze H.J.
      • Stoorvogel W.
      Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.
      ,
      • de Gassart A.
      • Geminard C.
      • Fevrier B.
      • Raposo G.
      • Vidal M.
      Lipid raft-associated protein sorting in exosomes.
      ) were present in the majority of our BCG phagosome preparations, including integrin β1 and β2; CD63; Rap-1B/Rap-1A; Rab-7; annexins A2, A4, and A5; Hsc70; Hsp90; actin; cofilin; tubulin; moesin; pyruvate kinase; enolase; elongation factor 1-α; and 14-3-3. The presence of integrins in the endocytic pathway has been reported previously (
      • Shakibaei M.
      • Zimmermann B.
      • Scheller M.
      Endocytosis of integrin alpha 5 beta 1 (fibronectin receptor) of mouse peritoneal macrophages in vitro: an immunoelectron microscopic study.
      ).

      Lipid Raft Proteins

      Lipid rafts and raft proteins (flotillin-1 and stomatin) are also enriched in the internal vesicles of MVBs and exosomes (
      • Février B.
      • Raposo G.
      Exosomes: endosomal-derived vesicles shipping extracellular messages.
      ,
      • de Gassart A.
      • Geminard C.
      • Fevrier B.
      • Raposo G.
      • Vidal M.
      Lipid raft-associated protein sorting in exosomes.
      ). We identified the raft proteins flotillin-1 and stomatin in all of our BCG phagosome preparations and in two of the three latex bead phagosomes (Table II). In addition to the lipid raft proteins flotillin and stomatin, we also identified the lipid raft protein erlin-2 in four of five BCG phagosome preparations but in none of the latex bead phagosomes (Table I). Although flotillin (
      • Dermine J.F.
      • Duclos S.
      • Garin J.
      • St-Louis F.
      • Rea S.
      • Parton R.G.
      • Desjardins M.
      Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes.
      ) and stomatin (
      • Snyers L.
      • Umlauf E.
      • Prohaska R.
      Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment.
      ) have been reported previously in association with phagocytic and endocytic pathways, erlin-2 was only recently identified as an ER-associated raft protein (
      • Browman D.T.
      • Resek M.E.
      • Zajchowski L.D.
      • Robbins S.M.
      Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER.
      ), and its presence in other intracellular compartments has not been reported.

      Signaling Proteins

      We identified many proteins involved in signaling by our proteomics analysis of the BCG phagosome. We identified ADP-ribosylation factor (Arf)-like protein 8b/8a (Arl 8b/8a) in the majority of BCG and latex bead phagosome preparations (four of five and two of three, respectively), although it was not detected in either of the two 5-day phagosome preparations (Table II and supplemental Table 1). Arf 8b colocalizes with late endosomes and lysosomes, interacts with tubulin, and is involved in motility of lysosomes (
      • Hofmann I.
      • Munro S.
      An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility.
      ). Thus, Arl 8b/8a may also be involved in motility of both BCG and latex bead phagosomes.
      We identified brain acid-soluble protein 1 (BASP1) in four of five BCG and all three latex bead phagosome preparations (Table II). BASP1, a myristoylated Ca2+-dependent calmodulin-binding protein that is phosphorylated by protein kinase C, associates with cholesterol-rich lipid rafts and, in neuronal cells, is thought to regulate actin interactions with the plasma membrane (
      • Maekawa S.
      • Iino S.
      • Miyata S.
      Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system.
      ). BASP1 has not been identified previously in association with phagosomes.
      Heterotrimeric G-proteins are membrane-associated proteins composed of α, β, and γ subunits that together function in numerous signal transduction pathways. We identified one or more G-protein subunits (e.g. guanine nucleotide-binding protein Gi, α-2 subunit (GNAI2), GNAI3, GNB1, GNG5, or GNG12) in the majority of our BCG and latex bead phagosome preparations, and we also identified the GNAI2 in our 1-day mouse J774 BCG phagosome preparation (supplemental Fig. 1 and Table 2). This protein, as well as other trimeric G-protein subunits, has also been identified in purified mouse J774 and Drosophila latex bead phagosomes (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ,
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ). Although the physiological role of G-proteins in mediating signal transduction events on the plasma membrane is well known (
      • Oldham W.M.
      • Hamm H.E.
      Heterotrimeric G protein activation by G-protein-coupled receptors.
      ), it is possible that they may also serve a similar function on the phagosomal membrane.
      We identified the Ras GTPase-activating-like protein IQGAP1 in all of our BCG phagosome preparations except for the 3-h phagosome preparation, but we did not detect this protein in any of the latex bead phagosome preparations (Table I). Similarly, IQGAP1 was not identified in prior reports in latex bead phagosomes isolated from mouse or Drosophila macrophages, suggesting a true difference between the BCG phagosome and the latex bead phagosome. This signal transduction protein participates in multiple cellular signaling pathways, including interaction with Rac1, Cdc42, and Ca2+/calmodulin to effect changes to the actin cytoskeleton (
      • Brown M.D.
      • Sacks D.B.
      IQGAP1 in cellular signaling: bridging the GAP.
      ).
      We identified six members of the 14-3-3 family (β/α, ε, η, γ, ζ/δ, and θ) in the BCG phagosome preparation at days 3–5 (with β/α, ζ/δ, and γ identified in all of the 3–5-day BCG phagosome preparations), but no members of the family were identified in the 3-h or 1-day BCG phagosome preparations, and no members of the family were identified in latex bead phagosome preparations until day 5 (supplemental Table 1), suggesting that this protein family may be recruited more abundantly to BCG phagosomes than to latex bead phagosomes in THP-1 cells. However, the 14-3-3 family proteins have been identified by proteomics in latex bead phagosomes purified from mouse J774 macrophages (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ). The 14-3-3 family proteins are involved in a large number of signaling pathways and interact with numerous proteins involved in vesicular transport (
      • Mrowiec T.
      • Schwappach B.
      14-3-3 proteins in membrane protein transport.
      ). Similarly, we identified adenylyl cyclase-associated protein 1 (CAP1) in all of the BCG phagosomes at 1–5 days and in the 5-day latex bead phagosome preparation. CAP1 binds actin monomers and directly regulates filament dynamics (
      • Hubberstey A.V.
      • Mottillo E.P.
      Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.
      ). Thus, CAP1 may regulate the interaction of BCG and latex bead phagosomes with actin microfilaments.

      Rab and Rab-related Proteins

      We identified many Rab GTPase and Rab-related proteins in both the BCG and latex bead phagosome preparations. We identified Rab-5C in all of our BCG and latex bead phagosome preparations (Table II). We have previously demonstrated the presence of Rab-5C on M. tuberculosis phagosomes by immunoelectron microscopy (
      • Clemens D.L.
      • Lee B.Y.
      • Horwitz M.A.
      Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate.
      ).
      We identified Rab-7A in four of our five BCG phagosome preparations and in all of our latex bead phagosome preparations (Table II). Rab-7A regulates vesicular traffic of late endosomes, and it has been detected on latex bead phagosomes of macrophages from mouse (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ) and Drosophila (
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ). We have previously demonstrated the presence of Rab-7 on the M. tuberculosis phagosome by immunoelectron microscopy (
      • Clemens D.L.
      • Lee B.Y.
      • Horwitz M.A.
      Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7.
      ).
      We identified Rab-11A/Rab-11B (Table II and supplemental Table 1) in all five of our BCG phagosome preparations and in two of our three latex bead phagosome preparations (all but day 5). Although Rab-11 is known for its role in endosomal recycling from a perinucleolar compartment to the plasma membrane, Rab-11 and its effector, Rab coupling protein, have been identified by immunofluorescence in patches on latex bead phagosomal membranes (
      • Damiani M.T.
      • Pavarotti M.
      • Leiva N.
      • Lindsay A.J.
      • McCaffrey M.W.
      • Colombo M.I.
      Rab coupling protein associates with phagosomes and regulates recycling from the phagosomal compartment.
      ) and by proteomics in purified mouse J774 latex bead phagosomes (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ). It has been proposed that Rab-11 may be involved in recycling of vesicles from the phagosome to the plasma membrane (
      • Damiani M.T.
      • Pavarotti M.
      • Leiva N.
      • Lindsay A.J.
      • McCaffrey M.W.
      • Colombo M.I.
      Rab coupling protein associates with phagosomes and regulates recycling from the phagosomal compartment.
      ). Although we observed a relative depletion of Rab-11 by Western immunoblotting in our phagosome preparations relative to its abundance in the PNS (Fig. 3B), Rab-11 may still be present and of functional importance on the phagosomal membrane.
      We identified Rab-1A/Rab-1B in four of our five BCG phagosome preparations and in the 3-day latex bead phagosome preparation (supplemental Table 1). Rab-1 also has been detected in latex bead phagosomes purified from Drosophila macrophages (
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ). Although Rab-1 is well known for regulation of transport between the ER-Golgi intermediate compartment and the Golgi compartment, Rab-1 has recently been demonstrated also to be involved in transport of vesicles from the ER-Golgi intermediate compartment toward the cell periphery (
      • Sannerud R.
      • Marie M.
      • Nizak C.
      • Dale H.A.
      • Pernet-Gallay K.
      • Perez F.
      • Goud B.
      • Saraste J.
      Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery.
      ). Thus, Rab-1 might also regulate the interaction of this ER-related compartment with the phagosome.
      We identified Rab-6A in four of the five BCG phagosome preparations but in none of the latex bead phagosome preparations (Table I). However, Rab-6A has been reported in latex bead phagosomes from Drosophila (
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ). Rab-6A regulates traffic within Golgi stacks and between ER and Golgi, but it has also been shown to play a role in cytokinesis and in a pathway that interacts with Rab-11 (
      • Miserey-Lenkei S.
      • Waharte F.
      • Boulet A.
      • Cuif M.H.
      • Tenza D.
      • El Marjou A.
      • Raposo G.
      • Salamero J.
      • Héliot L.
      • Goud B.
      • Monier S.
      Rab6-interacting protein 1 links Rab6 and Rab11 function.
      ). The role of Rab-6A on phagosomes is not known.
      We identified Rab-21 in four of five BCG phagosome preparations and in two of three latex bead phagosome preparations (Table II). Rab-21 was not reported in prior latex bead phagosome proteomics studies. Rab-21 is present on early endosomes (
      • Simpson J.C.
      • Griffiths G.
      • Wessling-Resnick M.
      • Fransen J.A.
      • Bennett H.
      • Jones A.T.
      A role for the small GTPase Rab21 in the early endocytic pathway.
      ) and has been shown to bind directly to integrins and to regulate the endocytic and exocytic traffic of integrins (
      • Pellinen T.
      • Arjonen A.
      • Vuoriluoto K.
      • Kallio K.
      • Fransen J.A.
      • Ivaska J.
      Small GTPase Rab21 regulates cell adhesion and controls endosomal traffic of beta1-integrins.
      ). We identified integrins as a component of the BCG phagosome, and thus Rab-21 may be involved in membrane trafficking of these molecules to or from the BCG phagosome.
      We identified Rab-14 in three of five BCG phagosome preparations but in none of the latex bead phagosome preparations (Table I). This finding is consistent with a report by Kyei et al. (
      • Kyei G.B.
      • Vergne I.
      • Chua J.
      • Roberts E.
      • Harris J.
      • Junutula J.R.
      • Deretic V.
      Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest.
      ), who found that Rab-14 was recruited to live but not dead M. bovis BCG phagosomes and that a dominant-negative form of Rab-14 promoted phagosomal maturation.
      We identified Ral-B/Ral-A (based on single peptide identifications that could be assigned to either protein) in three of our five BCG phagosome preparations, but we did not detect this Ras GTPase in any of our latex bead phagosome preparations (Table I). It has not been reported in prior proteomics studies of latex bead phagosomes (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ,
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ). Ral-B has been shown to regulate exocytosis (
      • Rossé C.
      • Hatzoglou A.
      • Parrini M.C.
      • White M.A.
      • Chavrier P.
      • Camonis J.
      RalB mobilizes the exocyst to drive cell migration.
      ,
      • Li G.
      • Han L.
      • Chou T.C.
      • Fujita Y.
      • Arunachalam L.
      • Xu A.
      • Wong A.
      • Chiew S.K.
      • Wan Q.
      • Wang L.
      • Sugita S.
      RalA and RalB function as the critical GTP sensors for GTP-dependent exocytosis.
      ), to play a role in organization of the actin cytoskeleton (
      • Oxford G.
      • Owens C.R.
      • Titus B.J.
      • Foreman T.L.
      • Herlevsen M.C.
      • Smith S.C.
      • Theodorescu D.
      RalA and RalB: antagonistic relatives in cancer cell migration.
      ), and to be involved in signal transduction of toll-like receptors by activation of NFκB via the IκB kinase Tank binding kinase 1 (TBK1) (
      • Chien Y.
      • Kim S.
      • Bumeister R.
      • Loo Y.M.
      • Kwon S.W.
      • Johnson C.L.
      • Balakireva M.G.
      • Romeo Y.
      • Kopelovich L.
      • Gale Jr., M.
      • Yeaman C.
      • Camonis J.H.
      • Zhao Y.
      • White M.A.
      RalB GTPase-mediated activation of the IkappaB family kinase TBK1 couples innate immune signaling to tumor cell survival.
      ). By immunofluorescence, we observed colocalization of endogenous Ral-B on the BCG phagosome.
      B.-Y. Lee, D. L. Clemens, and M. A. Horwitz, unpublished data.
      In macrophages in which we overexpressed recombinant GFP-Ral-B, we observed colocalization of Ral-B with both BCG and latex bead phagosomes.2 The function of Ral-B on the phagosomal membrane remains to be determined, but in view of its previously demonstrated roles, it could be important in regulating vesicular traffic or in signal transduction of toll-like receptors present on the phagosomal membrane.
      We identified Rap-1A/Rap-1B in all of the latex bead phagosome preparations and in three of the five BCG phagosome preparations (supplemental Table 1). Rap-1A/Rap-1B have been identified by immunomicroscopy on late endosomes/lysosomes and phagosomes (
      • Pizon V.
      • Desjardins M.
      • Bucci C.
      • Parton R.G.
      • Zerial M.
      Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex.
      ) and on exosomes (
      • Théry C.
      • Boussac M.
      • Véron P.
      • Ricciardi-Castagnoli P.
      • Raposo G.
      • Garin J.
      • Amigorena S.
      Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.
      ) and by proteomics studies on latex bead phagosomes from mouse macrophages (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ). Their function is not known, but there is evidence that Rap-1A has a role in initiation of the oxidative burst in neutrophils (
      • Li Y.
      • Yan J.
      • De P.
      • Chang H.C.
      • Yamauchi A.
      • Christopherson 2nd, K.W.
      • Paranavitana N.C.
      • Peng X.
      • Kim C.
      • Munugalavadla V.
      • Kapur R.
      • Chen H.
      • Shou W.
      • Stone J.C.
      • Kaplan M.H.
      • Dinauer M.C.
      • Durden D.L.
      • Quilliam L.A.
      Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b proteins.
      ) and that Rap-1B regulates integrin signaling (
      • Bernardi B.
      • Guidetti G.F.
      • Campus F.
      • Crittenden J.R.
      • Graybiel A.M.
      • Balduini C.
      • Torti M.
      The small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin alphaIIbbeta3.
      ).
      We identified Rab-guanine dissociation inhibitor (GDI) in two of five BCG phagosome preparations and also identified the Rho-GDP dissociation inhibitor in two of five BCG phagosome preparations but did not identify this protein in any of our latex bead phagosome preparations (supplemental Table 1). This finding complements the study of Fratti et al. (
      • Fratti R.A.
      • Chua J.
      • Deretic V.
      Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes.
      ), who reported persistence of GDI with BCG phagosomes but not latex bead phagosomes at early times after uptake and proposed that persistence of GDI on the mycobacterial phagosome was due to mycobacterial activation of p38 mitogen-activated protein kinase (MAPK) leading to phosphorylation of GDI. Greater levels of GDI on the BCG phagosome may displace EEA1 from the phagosomal membrane and impair phagosome maturation (
      • Fratti R.A.
      • Chua J.
      • Deretic V.
      Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes.
      ,
      • Cavalli V.
      • Vilbois F.
      • Corti M.
      • Marcote M.J.
      • Tamura K.
      • Karin M.
      • Arkinstall S.
      • Gruenberg J.
      The stress-induced MAP kinase p38 regulates endocytic trafficking via the GDI:Rab5 complex.
      ).

      Secreted Proteins

      Apolipoproteins D and E are 21-kDa proteins synthesized and secreted by macrophages that are involved in cholesterol transport and catabolism. We identified either apolipoprotein D or apolipoprotein E on four of five BCG phagosome preparations and on all three latex bead phagosome preparations (supplemental Table 1). Others have also identified apolipoproteins in mouse J774 latex bead phagosomes (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ). These proteins could be delivered to the BCG phagosome via either the endocytic or the secretory route. However, our mass spectrometry analysis of both apolipoproteins D and E indicated that they are of human rather than bovine origin, excluding the possibility that they were derived from the bovine serum in which the macrophages were cultured after the initial infection. Thus, it is likely that they are derived from the macrophage secretory pathway rather than of endocytic origin.
      Whereas we identified apolipoproteins D and/or E in both early and late BCG phagosome preparations, we identified complement factors C3 and C9 only on the 3-h latex bead and BCG phagosome preparations, respectively (supplemental Table 1). Their presence only early after phagocytosis likely reflects adsorption or fixation of complement to the beads or bacteria prior to uptake.
      We identified the secreted protein retinoid-inducible serine carboxypeptidase in three of five BCG phagosome preparations and in one latex bead phagosome preparation (supplemental Table 1). Kollman et al. (
      • Kollmann K.
      • Mutenda K.E.
      • Balleininger M.
      • Eckermann E.
      • von Figura K.
      • Schmidt B.
      • Lübke T.
      Identification of novel lysosomal matrix proteins by proteome analysis.
      ) have shown that retinoid-inducible serine carboxypeptidase is delivered to endosomes/lysosomes by the M6PR.

      Proteins of Unknown Function

      We identified several proteins of unknown function in our BCG and latex bead phagosome preparations. Three proteins of unknown function that we identified on the majority of our BCG phagosome preparations but on none of the latex bead phagosome preparations are FAM3C, tumor protein D54, and synaptic vesicle amine transport membrane protein-1 (VAT-1) homolog. We identified FAM3C and tumor protein D54 in four of five BCG phagosome preparations and the VAT-1 homolog in three of five BCG phagosome preparations, but we identified none of these proteins in any of our latex bead phagosome preparations (Table I). FAM3C, tumor protein D54, and the VAT-1 homolog also were not identified in prior mouse or Drosophila latex bead phagosome preparations (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
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      ,
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      A systems biology analysis of the Drosophila phagosome.
      ). We also identified FAM3C by immunofluorescence on the BCG phagosome. FAM3C has sequence similarity to human and mouse proteins from the cytokine-like gene family and has both a predicted signal sequence and a transmembrane domain (
      • Pilipenko V.V.
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      Genomic organization and expression analysis of the murine Fam3c gene.
      ). The function of FAM3C is unknown, and it is unclear whether it is a secreted or a membrane protein. Tumor protein D54 has been identified by MS-based proteomics studies in normal human astrocytes (
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      Heroin-Induces Differential Protein Expression by Normal Human Astrocytes (NHA).
      ), in breast tumor cells (
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      Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry.
      ), and in lipid droplets from lipolytically stimulated (but not unstimulated) adipocytes (
      • Brasaemle D.L.
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      • Shapiro L.
      • Wang R.
      Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.
      ). No studies of its physiological function have been reported. The synaptic vesicle membrane protein VAT-1 homolog is a 41-kDa integral membrane protein with sequence similarity to the zinc-containing alcohol dehydrogenase family (
      • Hayess K.
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      Mammalian protein homologous to VAT-1 of Torpedo californica: isolation from Ehrlich ascites tumor cells, biochemical characterization, and organization of its gene.
      ) and to the mammalian lens ξ-crystallin (
      • Persson B.
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      • Jörnvall H.
      A super-family of medium-chain dehydrogenases/reductases (MDR). Sub-lines including zeta-crystallin, alcohol and polyol dehydrogenases, quinone oxidoreductase enoyl reductases, VAT-1 and other proteins.
      ). It has been identified in human and mouse epithelial cells and shown to have ATPase activity and to be calcium-regulated (
      • Hayess K.
      • Kraft R.
      • Sachsinger J.
      • Janke J.
      • Beckmann G.
      • Rohde K.
      • Jandrig B.
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      Mammalian protein homologous to VAT-1 of Torpedo californica: isolation from Ehrlich ascites tumor cells, biochemical characterization, and organization of its gene.
      ,
      • Koch J.
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      • Schaefer B.M.
      Human VAT-1: a calcium-regulated activation marker of human epithelial cells.
      ), but its function is otherwise unknown.

      Evaluation of Flotillin-1 Location by Immunofluorescence

      Our proteomics analysis detected flotillin-1 in all of our BCG phagosome preparations and also in two of our latex bead phagosome preparations. Nevertheless, our Western immunoblots (Fig. 4) demonstrated that flotillin-1 was depleted in the BCG phagosome preparation relative to the latex bead phagosome preparation and the PNS. To evaluate further the association of flotillin-1 with mycobacterial phagosomes, we examined the degree of colocalization of flotillin-1 with M. bovis BCG and M. tuberculosis phagosomes in THP-1 cells. In accord with our Western immunoblotting data, M. bovis BCG phagosomes exhibited lower levels of flotillin-1 fluorescence than latex bead phagosomes at 3 days postinfection, although some heterogeneity in intensity of staining was observed (data not shown). To determine whether the heterogeneity in flotillin-1 immunofluorescence correlated with mycobacterial metabolic activity, we infected THP-1 macrophages with a recombinant M. tuberculosis (Mtb-iGFP) whose GFP expression is induced in response to IPTG (
      • Lee B.Y.
      • Clemens D.L.
      • Horwitz M.A.
      The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages.
      ) and examined GFP expression and flotillin-1 distribution 48 h postinfection. Only M. tuberculosis that are alive and metabolically active are able to express GFP in response to IPTG, which is added after infection of the macrophages. Using this system, we observed that Mtb-iGFP that fail to express GFP in response to IPTG reside within acidified compartments that fuse with Texas Red-dextran-labeled lysosomes, whereas Mtb-iGFP that do express GFP in response to IPTG reside in non-acidified compartments that do not fuse with Texas Red-dextran (
      • Lee B.Y.
      • Clemens D.L.
      • Horwitz M.A.
      The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages.
      ). In the current study, we observed an inverse correlation between GFP expression by the Mtb-iGFP and flotillin-1 immunofluorescence around the bacteria (Fig. 5, A and B). In contrast, killed M. tuberculosis and latex beads colocalized consistently with flotillin-1 immunofluorescence (Fig. 5B).
      Figure thumbnail gr5
      Fig. 5Flotillin-1 immunofluorescence on M. tuberculosis phagosomes in THP-1 cells correlates inversely with metabolic activity of mycobacteria. A, metabolic activity of Mtb-iGFP was assessed by addition of IPTG after infection and visualization of green fluorescent protein expression at fixation 48 h postinfection (a and d), and Mtb-iGFP, independent of metabolic status, was visualized by blue fluorescence with aminomethylcoumarin-labeled anti-LAM antibody (a and d). Flotillin-1 distribution was visualized by staining with Texas Red-labeled anti-human flotillin-1 antibody (b and e). The merged color images are shown on the right (c and f). Metabolically inactive Mtb-iGFP (bacteria that do not express GFP after IPTG induction; a–f, arrows) consistently colocalized with flotillin-1 labeled with Texas Red (arrows indicating three bacteria in b and one bacterium in e). The metabolically active Mtb-iGFP (a–c, arrowheads) showed weaker and less consistent colocalization with flotillin-1 (b). B, quantitative assessment of flotillin-1 immunofluorescence demonstrates relatively low levels of colocalization of flotillin-1 on metabolically active Mtb-iGFP (GFP+ M. tuberculosis) compared with much higher levels of colocalization with inactive Mtb-iGFP (GFP− M. tuberculosis), formalin-killed Mtb-GFP (Killed M. tuberculosis), and latex beads at 48 h postinfection (and 46.5 h post-IPTG induction) in THP-1 cells. The experiment was performed twice with similar results. Values shown are means and error bars indicate S.D. for duplicate determinations for at least 40 bacteria or beads.

      DISCUSSION

      We demonstrated a method for high level purification of BCG phagosomes by use of a combination of differential centrifugation and density gradient sedimentation on sucrose and iodixanol gradient media that is amenable to further molecular studies of the M. bovis BCG and M. tuberculosis phagosomes. We consistently observed a decrease in buoyant density of BCG and M. tuberculosis over time with growth in macrophages. The change in density most likely reflects a change in the density of the mycobacteria rather than a change in the macrophage contribution to the phagosome because electron microscopy analysis revealed a mixture of free bacteria (non-membrane-bound) as well as membrane-bound bacteria in the same density fractions. Moreover, phagosomes containing killed bacteria exhibited a heavier density than those containing live bacteria that have grown in macrophages for 1 or more days. The decrease in mycobacterial density probably reflects a change in lipid composition of the mycobacteria or an increase in the ratio of mycobacterial lipid to protein and carbohydrate that is induced by growth in macrophages.
      Our proteomics analysis is an extremely sensitive method for study of the composition of the BCG phagosome and represents a first stage in the identification of proteins associated with the mycobacterial phagosome. An important strength of the technique is that it provides an unbiased assessment of the proteins present. However, confirmation of the presence of proteins identified by proteomics should be obtained by independent techniques such as immunofluorescence microscopy of intact macrophages infected with pathogenic mycobacteria. Hence, the list of proteins identified herein should be considered a list of candidate proteins present on the mycobacterial phagosome. Some of the proteins identified may reflect contaminants rather than proteins that are truly associated with the phagosome. Furthermore, although the proteins present on latex bead phagosomes and BCG phagosomes show considerable overlap with relatively few consistently detected proteins on only one or the other phagosome, the abundance of various proteins may differ greatly for latex bead and BCG phagosomes. For example, we identified flotillin-1 and the vATPase by mass spectrometry at all time points on both the latex bead and the BCG phagosome; however, by Western immunoblotting of phagosomes isolated 3 days postinfection, flotillin-1 and vATPase were enriched on the latex bead phagosomes but relatively scarce on the BCG phagosome. Consistent with this, by fluorescence microscopy, we observed weaker flotillin-1 immunofluorescence on BCG and M. tuberculosis phagosomes than on latex bead phagosomes, and use of an IPTG-inducible expression system showed that flotillin-1 colocalization with M. tuberculosis varied inversely with the metabolic activity of the mycobacteria. Flotillin-1 has been shown to be recruited to maturing latex bead phagosomes (
      • Dermine J.F.
      • Duclos S.
      • Garin J.
      • St-Louis F.
      • Rea S.
      • Parton R.G.
      • Desjardins M.
      Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes.
      ) and to be a constituent of lipid rafts that also contain other proteins important to membrane trafficking and phagosome formation, including the α- and β-subunits of heterotrimeric G proteins as well as subunits of the proton pump vATPase (
      • Li N.
      • Mak A.
      • Richards D.P.
      • Naber C.
      • Keller B.O.
      • Li L.
      • Shaw A.R.
      Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation.
      ). Flotillin-1 may act as a scaffolding protein to organize lipid rafts containing the vATPase for delivery to the maturing phagosome. Thus, the relative scarcity of flotillin-1 on the BCG phagosome, as indicated in our Western immunoblotting and immunofluorescence studies, may be important in excluding the vATPase and resisting acidification.
      Consistent with prior mass spectrometry-based proteomics analyses of phagosomes, we found multiple ER-associated proteins (including calnexin, endoplasmin, and protein-disulfide isomerase) in all of our latex bead and BCG phagosomal preparations. This could reflect a contribution of the ER to formation of the phagosome, or it may reflect non-ER functions of these proteins. For example, protein-disulfide isomerase A3 (Erp57), which we detected in all of our phagosome preparations, although generally considered an ER protein, has been shown also to reside in the cytosol and to form a complex with the cytosolic signaling protein STAT3 (
      • Turano C.
      • Coppari S.
      • Altieri F.
      • Ferraro A.
      Proteins of the PDI family: unpredicted non-ER locations and functions.
      ). Protein-disulfide isomerase, also detected in all of our phagosome preparations, has been shown to be present at high levels on the plasma membrane of lymphoid cells (
      • Turano C.
      • Coppari S.
      • Altieri F.
      • Ferraro A.
      Proteins of the PDI family: unpredicted non-ER locations and functions.
      ).
      Specific recruitment of components of the ER to a variety of phagosomes (including those containing latex beads, zymosan, killed bacteria, and T. gondii) has been reported by numerous investigators based on studies of isolated phagosomes, immunoelectron microscopy, flow organellometry, and antigen processing and presentation (
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      The phagosome proteome: insight into phagosome functions.
      ,
      • Gagnon E.
      • Duclos S.
      • Rondeau C.
      • Chevet E.
      • Cameron P.H.
      • Steele-Mortimer O.
      • Paiement J.
      • Bergeron J.J.
      • Desjardins M.
      Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages.
      ,
      • Giodini A.
      • Rahner C.
      • Cresswell P.
      Receptor-mediated phagocytosis elicits cross-presentation in nonprofessional antigen-presenting cells.
      ,
      • Ackerman A.L.
      • Kyritsis C.
      • Tampé R.
      • Cresswell P.
      Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.
      ,
      • Goldszmid R.S.
      • Coppens I.
      • Lev A.
      • Caspar P.
      • Mellman I.
      • Sher A.
      Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii-infected dendritic cells.
      ,
      • Stuart L.M.
      • Boulais J.
      • Charriere G.M.
      • Hennessy E.J.
      • Brunet S.
      • Jutras I.
      • Goyette G.
      • Rondeau C.
      • Letarte S.
      • Huang H.
      • Ye P.
      • Morales F.
      • Kocks C.
      • Bader J.S.
      • Desjardins M.
      • Ezekowitz R.A.
      A systems biology analysis of the Drosophila phagosome.
      ,
      • Houde M.
      • Bertholet S.
      • Gagnon E.
      • Brunet S.
      • Goyette G.
      • Laplante A.
      • Princiotta M.F.
      • Thibault P.
      • Sacks D.
      • Desjardins M.
      Phagosomes are competent organelles for antigen cross-presentation.
      ,
      • Desjardins M.
      ER-mediated phagocytosis: a new membrane for new functions.
      ). Most pertinent is the report by Grotzke et al. (
      • Grotzke J.E.
      • Harriff M.J.
      • Siler A.C.
      • Nolt D.
      • Delepine J.
      • Lewinsohn D.A.
      • Lewinsohn D.M.
      The Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle.
      ) of a specific association of ER components (including transporter associated with antigen processing (TAP) and protein-disulfide isomerase) with the M. tuberculosis phagosome based on flow organellometry and antigen processing and presentation studies.
      The identification of numerous proteins that were found exclusively on either the BCG or latex bead phagosome, differentially expressed on one of these phagosomes relative to the other, or present at different times after phagocytosis sets the stage for further studies aimed at precisely delineating the pathway of pathogenic mycobacteria in host macrophages and the interaction of cellular organelles, macromolecular complexes, signaling proteins, trafficking proteins, enzymes, and other cellular molecules with the mycobacterial phagosome. The phagosome isolation technique described herein also allows for future studies of non-protein constituents of the mycobacterial phagosome, further elucidating the interaction of the phagosome with host cell organelles and other molecular constituents.

      Acknowledgments

      We are grateful to Matt Schibler for assistance with confocal microscopy and to Melissa Taylor for expert technical assistance.

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