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Study of Phagolysosome Biogenesis in Live Macrophages
Published on: March 10, 2014
Phagocytic processing of the macrophage endoparasite, Mycobacterium avium, in comparison to phagosomes which contain
C de Chastellier1, T Lang, L Thilo
1INSERM U 411, Laboratoire de Microbiologie, UFR de Médecine Necker-Enfants Malades, Paris/France.
Abstract:
The intraphagosomal survival strategy of pathogenic mycobacteria was studied in bone marrow-derived mouse macrophages. These bacteria survive inside phagosomes by interfering in an unknown manner with phagosome processing which normally would lead to digestion of the phagocytic particle in phagolysosomes. Here, phagosome processing was compared for different phagocytic particles: live Mycobacterium avium, degradable Bacillus subtilis, or indigestible latex beads. We show detailed electron microscopic morphological observations which characterize various phases of interaction between endocytic organelles and phagosomes. We measured fusion of phagosomes with early endosomes or with lysosomes by using newly internalized endocytic contents (horseradish peroxidase, HRP) and membrane marker (plasma membrane glycoconjugates labeled with [3H]galactose via exoglycosylation). Morphometric analysis of these observations showed that the nature of the phagocytic particle affects phagosome processing: As long as particles remain undigested, maturation of phagosomes is prevented and they remain fusogenic towards early endosomes; concurrent to particle digestion, phagosome processing proceeds towards transfer of phagocytic contents to phagolysosomes which display kinetic and compositional characteristics of lysosomes. As an intact phagocytic particle, M. avium remains in non-matured phagosomes which fuse with early endosomes, but not with lysosomes. Fusion with early endosomes is reduced, thereby indicating the stage where this endoparasite exerts its effect.
Insights
Pathogenic mycobacteria, like Mycobacterium avium, survive in macrophages by blocking phagosome maturation. This study reveals how bacteria prevent phagosome fusion with lysosomes, a key survival strategy.
Area of Science:
- Cell Biology
- Microbiology
- Immunology
Background:
- Pathogenic mycobacteria evade host immune defenses by surviving within macrophages.
- Intraphagosomal survival involves interfering with phagosome processing and preventing lysosomal digestion.
Purpose of the Study:
- To investigate the intraphagosomal survival strategy of pathogenic mycobacteria in mouse macrophages.
- To compare phagosome processing dynamics with different phagocytic particles: Mycobacterium avium, Bacillus subtilis, and latex beads.
Main Methods:
- Detailed electron microscopy to observe interactions between endocytic organelles and phagosomes.
- Quantification of phagosome fusion with early endosomes and lysosomes using horseradish peroxidase and radiolabeled membrane markers.
- Morphometric analysis to assess the impact of particle nature on phagosome maturation.
Main Results:
- Phagosome maturation is inhibited when undigested particles are present, maintaining fusion with early endosomes.
- Digestion of particles correlates with phagosome maturation and fusion with lysosomes.
- Mycobacterium avium, as an undigested particle, resides in non-matured phagosomes with reduced fusion to lysosomes, indicating interference with early endosome fusion.
Conclusions:
- The nature of the phagocytic particle critically influences phagosome processing and maturation.
- Mycobacterium avium actively interferes with phagosome maturation, specifically by modulating fusion events, to ensure its intraphagosomal survival.
