title: Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.
abstract: Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.
authors.affiliation: Department of Pharmaceutics, State University of New York at Buffalo, Amherst 14260, USA.
authors.forename: Y K
authors.initials: YK
authors.lastname: Oh
authors.forename: R M
authors.initials: RM
authors.lastname: Straubinger
mesh_terms: D000818:Animals; D002460:Cell Line; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000502:physiology*; D008264:Macrophages / Q000382:microbiology*; D008561:Membrane Fusion; D051379:Mice; D018715:Microscopy, Video; D009162:Mycobacterium avium / Q000254:growth & development / Q000276:immunology*; D009895:Opsonin Proteins; D010588:Phagosomes / Q000502:physiology*; D012235:Rhodamines; D012486:Salmonella typhimurium / Q000276:immunology; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D009895:Opsonin Proteins; D012235:Rhodamines