title: Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria.
abstract: Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.
authors.affiliation: Cell Biology Program, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.
authors.forename: Otilia V
authors.initials: OV
authors.lastname: Vieira
authors.forename: Rene E
authors.initials: RE
authors.lastname: Harrison
authors.forename: Cameron C
authors.initials: CC
authors.lastname: Scott
authors.forename: Harald
authors.initials: H
authors.lastname: Stenmark
authors.forename: David
authors.initials: D
authors.lastname: Alexander
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Jean
authors.initials: J
authors.lastname: Gruenberg
authors.forename: Alan D
authors.initials: AD
authors.lastname: Schreiber
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D056827:Endosomal Sorting Complexes Required for Transport; D015870:Gene Expression; D051379:Mice; D009154:Mutation; D019910:Mycobacterium marinum / Q000472:pathogenicity*; D020102:Mycobacterium smegmatis / Q000472:pathogenicity*; D010587:Phagocytosis; D010588:Phagosomes / Q000382:microbiology* / Q000502:physiology*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014774:Virulence; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D000077191:Wortmannin