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Isolation of Bead Phagosomes to Study Virulence Function of M. tuberculosis Cell Wall Lipids

  • Anna C. Geffken
  • Emmanuel C. Patin
  • Ulrich E. SchaibleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)

Abstract

Following pathogen recognition by macrophages, the causative agent of human tuberculosis, Mycobacterium tuberculosis, is internalized by receptor-mediated phagocytosis. Phagosomes containing nonpathogenic bacteria usually follow a stepwise maturation process to phagolysosomes where bacteria are eliminated. However, as a hallmark of M. tuberculosis virulence, pathogenic mycobacteria inhibit phagosome maturation in order to generate an intracellular niche for persistence and replication in resting macrophages. In contrast, activation by interferon gamma and tumor necrosis alpha activates microbicidal effectors of macrophages such as nitric oxide synthase, NO-mediated apoptosis and LRG-47-linked autophagy, which drives M. tuberculosis into phagolysosomes. Glycolipid compounds of the mycobacterial cell wall have been suggested as virulence factors and several studies revealed their contribution to mycobacterial interference with phagosome maturation. To study their effect on phagosome maturation and to characterize phagosomal protein and lipid compositions, we developed a reductionist mycobacterial lipid-coated bead model. Here, we provide protocols to “infect” macrophages with lipid-coated magnetic beads for subsequent purification and characterization of bead phagosomes. This model has been successfully employed to characterize the virulence properties of trehalose dimycolate, as one of the cell wall glycolipids essential for inhibition of phagosome maturation.

Key words

Phagocyte Macrophage Phagosome Lipid Bead Proteome 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anna C. Geffken
    • 1
  • Emmanuel C. Patin
    • 1
    • 2
  • Ulrich E. Schaible
    • 1
    • 2
    • 3
    Email author
  1. 1.Cellular Microbiology, Priority Area InfectionsResearch Center BorstelBorstelGermany
  2. 2.Department of ImmunologyLondon School of Hygiene and Tropical MedicineLondonUK
  3. 3.German Centre of Infection Research, TTU-TBBorstelGermany

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