The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome

Abstract

Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Gué rin (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. Molecular & Cellular Proteomics 9:32-53, 2010.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 (1-5). 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, 1 and LAMP-2 and the lysosomal acid protease cathepsin D (6 -10). 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...

Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion

“…The results of studies on the phagolysosomal proteome vary depending on different factors: (i) the usage of cell lines or primary cells (23), (ii) the ingested particles e.g. latex beads, apoptotic cells, pathogen-associated structures or pathogens (16)(17)(18)22), (iii) the age of the phagolysosome (15,17), (iv) the analyzed fraction e.g. entire phagolysosome or membrane domains (detergent-resistant or detergent-soluble membranes) (15) and (v) the activation status of the cells, i.e.…”

“…For several pathogenic microorganisms, proteomic studies unraveled the specific protein composition of phagolysosomes for a better understanding of the phagosomal maturation process and its modulation by pathogens (15)(16)(17)(18)(19)(20)(21)(22)(23). These studies demonstrated that the protein composition of the phagosome is highly dynamic, specific for the ingested particle, depends on the stage of phagosomal maturation, the type or cell line and the activation status of the phagocyte.…”

Phagosome proteomics: A powerful tool to assess bacteria-mediated immunomodulation

“…18 Jutras et al found that γ-secretase is a functional phagosome component that resides on newly formed phagosomes and remains associated with them throughout their maturation into phagolysosomes. 19 With proteomics, Nicastrin was found to be present on BCG-and latex-bead-containing phagosomes in human THP-1 macrophages 20 as well as on phagosomes from Mtb infected mouse bone-marrow-derived macrophages. 21 Nicastrin is a member of the γ-secretase that is an integral membrane-bound protease complex.…”

“…Lee et al used radio-isotope labeled whole cell lysates that never saw latex bead or bacterial particles as an internal standard to estimate the degree of contamination from other organelles during a phagosome preparation. 20 The approach estimated the number of contaminating protein spots on a 2D gel but did not provide a quantitation of the contaminating fraction of individual proteins. However, it can be adapted for using stable-isotope labeling instead of radioactive labeling of host cells to estimate the contaminating fraction of of bacteria-mediated immunomodulation pathways.…”

Proteomic progress in studying tuberculosis from 2010 to 2011

“…Proteins involved in membrane trafficking and signal transduction such as Ras GTPase-activating-like protein IQGAP1 were detected to be regulated through a proteomic study [28]. In this study, the phagosomes from BCGinfected human macrophages were purified, and 447 human host proteins were identified.…”