Trehalose dimycolate interferes with FcγR-mediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling.

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6'-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5' phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.

Purification and proteomics of pathogen-modified vacuoles and membranes.

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

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

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.