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

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.

Purification and proteomics of pathogen-modified vacuoles and membranes

“…To characterize the virulence function of purified mycobacterial cell wall lipids in Mtb phagosome biogenesis, a simplified glycolipid-coated bead model was employed (Axelrod et al, 2008 ; Geffken et al, 2015 ). This approach differs from the protocols described above for analysis of other pathogen-containing vacuoles.…”

“…To achieve this task, we used a comparative proteomics approach analyzing purified phagosomes containing either control or TDM-coated beads. The procedure to purify bead phagosomes from macrophages has been described in detail (Geffken et al, 2015 ) (Figure 3B ). Briefly, RAW 264.7 macrophages were “bead-infected” using dynabeads covalently coated with BSA and TDM, incubated for 30 min at 37°C and scraped into ice-cold PBS.…”

“…RAW 264.7 MO were infected with TDM-coated beads, lysed after 30 min, and bead-phagosomes were purified (Geffken et al, 2015 ). The dynabead-phagosome preparation was subjected to LC-MS/MS and analyzed via Sequest and Scaffold 4.…”

“…We recently described the role of WASH-mediated actin recruitment to the mycobacterial phagosome as a prerequisite for inhibition of phagosome maturation and intracellular bacterial growth (Kolonko et al, 2014 ). Taken together, our improved purification protocol (Geffken et al, 2015 ), combined with high-resolution MS-based proteomics, revealed candidates putatively involved in TDM-bead phagosome formation, and therefore, most likely play a role for Mtb interactions within host MO (Kolonko et al, 2014 ).…”