Incorporation ofMycobacterium tuberculosisLipoarabinomannan into Macrophage Membrane Rafts Is a Prerequisite for the Phagosomal Maturation Block

Abstract

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannosecapped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.Mycobacterium tuberculosis, the etiological agent of tuberculosis, spreads by aerosol, mainly infecting alveolar macrophages, by which the bacterium is ingested (17). Through inhibition of macrophage functions, the bacterium modulates the host immune response (25). The best-characterized virulence factor of M. tuberculosis, lipoarabinomannan (LAM), is an abundant glycolipid, which is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and extends through the cell wall of the bacterium (5). During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage via specific receptors, including the macrophage mannose receptor and complement receptor 3 (13,14,19), and can then be detected at multiple sites in the cell (27). The host cell exports LAM from the phagosome in an exocytosis-like manner, eliciting responses in bystander cells (1-3). The M. tuberculosis surface harbors mannose-capped LAM (ManLAM), whereas other, less pathogenic, mycobacteria contain LAM either with a phospho-myo-inositol cap (PILAM) or no cap (6). The type of capping and the presence of the GPI anchor is crucial for virulence (13,14). The effects of Man-LAM on the host cell are multiple, but the interference of ManLAM with phagosomal maturation is the best characterized (21) and has been demonstrated in murine and human macrophages (10,12,14).Studies on human lymphocytes showed that LAM localizes to membrane rafts of the lymphocyte membrane, thereby interfering with signaling affecting cytokine production (20).Membrane rafts are cholesterol-and glycosphingolipid-rich domains that act as platforms for cell signaling processes (16). The aim of this study was to investigate whether the effects of LAM are due to incorporation of LAM into membrane rafts of human macrophages. We establish that LAM is incorporated into membrane rafts of the macrophage cell membrane via its GPI anchor, resulting in reduced phagosomal maturation. MATERIALS AND METHODSAntigens and antibodies. ManLAM, cell wall fractions (CWF), and phosphatidylinositol mannosides (PIM) from the virulen...

Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

“…In addition to Leishmania and Brucella mentioned above, Mycobacterium tuberculosis also appears to inhibit the assembly of these structures shortly after its internalization (13,14,60). In the case of Leishmania, it was shown that this parasite alters several of the functional properties of phagosomes, including the ability of this organelle to fuse with late endosomes and lysosomes (61).…”

The innate immune response in human tuberculosis

“…After phagocytosis, Mtb blocks phagosome acidification as well acquisition of hydrolytic enzymes and anti‐microbial peptides. Two major Mtb virulence factors are involved in the blockage of the phagosomal maturation in human cell lines: the glycolipid lipoarabinomannan (LAM) (Hmama et al ., 2004; Kang et al ., 2005; Welin et al ., 2008) and the secreted tyrosine phosphatase (PtpA) (Bach et al ., 2008; Wong et al ., 2011; Wong and Jacobs, 2011). Mtb lacking the surface lipid trehalose dimycolate (TDM) failed to block phagosome maturation in mouse macrophages (Katti et al ., 2008) but to date, this has not been shown in human cells.…”

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts

“…Human monocyte-derived macrophages were isolated from heparinised donor blood as previously described [14]. The cells were differentiated in Macrophage SFM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 4 mM L-glutamine (Life Technologies …”

“…The lipid anchor of LPG is characterized by an extended, saturated fatty acid residue [13], suggesting that it may be intercalated into host cell detergent-resistant membranes, DRM. We have recently shown that lipoarabinomannan (LAM) from Mycobacterium tuberculosis, whose molecular structure is reminiscent of LPG, is inserted into the membrane rafts of the host cell [14]. From this platform, LAM is able to delay phagosomal maturation, thereby being beneficial for M. tuberculosis virulence.…”