title: The Mycobacterium tuberculosis phagosome in human macrophages is isolated from the host cell cytoplasm.
abstract: Knowledge of whether Mycobacterium tuberculosis resides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy. To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) to M. tuberculosis within human macrophages. We infected the macrophages with wild-type or green fluorescent protein-expressing M. tuberculosis, microinjected Fab fragments directed against a major surface antigen of M. tuberculosis into the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy. Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments against M. tuberculosis did not stain the bacterium within its phagosome. In contrast, microinjected Fab fragments against Listeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium. Our study shows that M. tuberculosis resides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, Center for Health Sciences, Los Angeles, California 90095, USA. dclemens@mednet.ucla.edu
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Clemens
authors.forename: Bai-Yu
authors.initials: BY
authors.lastname: Lee
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Horwitz
mesh_terms: D000907:Antibodies, Bacterial / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018274:Electroporation; D049452:Green Fluorescent Proteins; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology* / Q000648:ultrastructure; D008845:Microinjections; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000472:pathogenicity*; D010588:Phagosomes / Q000382:microbiology* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins