title: Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2.
abstract: Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Binker
authors.forename: Laura I
authors.initials: LI
authors.lastname: Cosen-Binker
authors.forename: Mauricio R
authors.initials: MR
authors.lastname: Terebiznik
authors.forename: Gustavo V
authors.initials: GV
authors.lastname: Mallo
authors.forename: Shannon E
authors.initials: SE
authors.lastname: McCaw
authors.forename: Eeva-Liisa
authors.initials: EL
authors.lastname: Eskelinen
authors.forename: Marion
authors.initials: M
authors.lastname: Willenborg
authors.forename: John H
authors.initials: JH
authors.lastname: Brumell
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
authors.forename: Scott D
authors.initials: SD
authors.lastname: Gray-Owen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009344:Neisseria gonorrhoeae / Q000378:metabolism / Q000472:pathogenicity; D010587:Phagocytosis / Q000502:physiology; D010588:Phagosomes / Q000378:metabolism / Q000382:microbiology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D060149:Tetraspanin 30; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D015415:Biomarkers; D002272:Carcinoembryonic Antigen; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D060149:Tetraspanin 30; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C554586:CD63 protein, human; C075729:CEACAM3 protein, human; C554587:Cd63 protein, mouse; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C497811:LAMP2 protein, human; C052255:Lamp1 protein, mouse; C423317:RILP protein, human; C000716463:rab7 GTP-binding proteins, human; C000716465:rab7 GTP-binding proteins, mouse