Lipoarabinomannan binding to lactosylceramide in lipid rafts is essential for the phagocytosis of mycobacteria by human neutrophils

Mycobacterial-specific glycolipids determine pathogenesis after the mycobacteria are engulfed by human neutrophils. Escaping the lysosomal death path Macrophages and neutrophils bind to and internalize microbes within endocytic organelles called phagosomes. Fusion of phagosomes with lysosomes destroys the microbe. Pathogenic mycobacteria, such as Mycobacterium tuberculosis, prevent phagosomal maturation and lysosomal fusion and thus survive and replicate within the host cell. Nakayama et al. found that mycobacterial membrane glycolipids called lipoarabinomannans (LAMs) of both pathogenic and nonpathogenic strains of mycobacteria were similarly efficient at binding to lactosylceramide (LacCer) in the plasma membrane lipid rafts of human neutrophils. Both types of mycobacteria were internalized into phagosomes. However, whereas the LAMs of nonpathogenic mycobacteria resulted in the colocalization of LacCer with the Src family kinase Hck in the phagosomal membrane, the activation of Hck, and maturation of the phagosomes, the LAMs of pathogenic mycobacteria failed to induce LacCer-Hck clustering, thus preventing phagolysosome formation. These data suggest that targeting the LAMs of pathogenic mycobacteria may enhance the innate immune response to these pathogens, which could be beneficial in treating tuberculosis. Pathogenic mycobacteria use virulence factors, including mannose-capped lipoarabinomannan (ManLAM), to survive in host phagocytic cells, such as neutrophils. We assessed the roles of lactosylceramide (LacCer, CDw17)–enriched lipid rafts in the phagocytosis of mycobacteria by human neutrophils and in the intracellular fate of phagocytosed mycobacteria. We showed that the association of the Src family kinase (SFK) Lyn with C24 fatty acid chain–containing LacCer was essential for the phagocytosis of mycobacteria by neutrophils. Assays with LacCer-containing liposomes, LacCer-coated plastic plates, and LAM-coated beads demonstrated that the phagocytosis of mycobacteria was mediated through the binding of LacCer to LAM. Both ManLAM from pathogenic species and phosphoinositol-capped LAM (PILAM) from nonpathogenic Mycobacterium smegmatis bound equivalently to LacCer to stimulate phagocytosis. However, PILAM from an M. smegmatis α1,2-mannosyltransferase deletion mutant (ΔMSMEG_4247), lacking the α1,2-monomannose side branches of the LAM mannan core, did not bind to LacCer or induce phagocytosis. An anti-LacCer antibody immunoprecipitated the SFK Hck from the phagosomes of neutrophils that internalized nonpathogenic mycobacteria but not from those that internalized pathogenic mycobacteria. Furthermore, knockdown of Hck by short inhibitory RNA abolished the fusion of lysosomes with phagosomes containing nonpathogenic mycobacteria. Further analysis showed that ManLAM, but not PILAM, inhibited the association of Hck with LacCer-enriched lipid rafts in phagosomal membranes, effectively blocking phagolysosome formation. Together, these findings suggest that pathogenic mycobacteria use ManLAM not only for binding to LacCer-enriched lipid rafts and entering neutrophils but also for disrupting signaling through Hck-coupled, LacCer-enriched lipid rafts and preventing phagolysosome formation.