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

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 alpha 1,2-mannosyltransferase deletion mutant (Delta MSMEG_4247), lacking the alpha 1,2-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.