title: Lipoarabinomannan binding to lactosylceramide in lipid rafts is essential for the phagocytosis of mycobacteria by human neutrophils.
abstract: 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.
authors.affiliation: Laboratory of Biochemistry, Juntendo University Faculty of Health Care and Nursing, Urayasu, Chiba 279-0023, Japan. Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakayama
authors.affiliation: Department of Anatomy, Juntendo University Faculty of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Hidetake
authors.initials: H
authors.lastname: Kurihara
authors.affiliation: Department of Microbiology, University of Massachusetts, Amherst, MA 01003-9364, USA. Department of Immunoregulation, Research Institute for Microbial Diseases, World Premier International Research Center Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Yasu S
authors.initials: YS
authors.lastname: Morita
authors.affiliation: Department of Immunoregulation, Research Institute for Microbial Diseases, World Premier International Research Center Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Taroh
authors.initials: T
authors.lastname: Kinoshita
authors.affiliation: Department of Medical Biotechnology and Translational Medicine, Interdisciplinary Laboratory for Advanced Technologies, University of Milan, Via Fratelli Cervi, Milano 20129, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Mauri
authors.affiliation: Department of Medical Biotechnology and Translational Medicine, Interdisciplinary Laboratory for Advanced Technologies, University of Milan, Via Fratelli Cervi, Milano 20129, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Prinetti
authors.affiliation: Department of Medical Biotechnology and Translational Medicine, Interdisciplinary Laboratory for Advanced Technologies, University of Milan, Via Fratelli Cervi, Milano 20129, Italy.
authors.forename: Sandro
authors.initials: S
authors.lastname: Sonnino
authors.affiliation: Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Yokoyama
authors.affiliation: Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Takamori
authors.affiliation: Laboratory of Biochemistry, Juntendo University Faculty of Health Care and Nursing, Urayasu, Chiba 279-0023, Japan. Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu, Chiba 279-0021, Japan. Infection Control Nursing, Juntendo University Graduate School of Health Care and Nursing, Urayasu, Chiba 279-0023, Japan. iwabuchi@juntendo.ac.jp.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Iwabuchi
mesh_terms: D015703:Antigens, CD / Q000276:immunology*; D006801:Humans; D007790:Lactosylceramides / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D021962:Membrane Microdomains / Q000276:immunology*; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D007790:Lactosylceramides; D008070:Lipopolysaccharides; C050016:lipoarabinomannan; C009744:CDw17 antigen