Isolation of bead phagosomes to study virulence function of M. tuberculosis cell wall lipids

Methods in Molecular Biology
Anna C GeffkenUlrich E Schaible

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 purificatio...Continue Reading

Citations

Jun 18, 2015·Frontiers in Cellular and Infection Microbiology·Jo-Ana HerwegHubert Hilbi

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