Related Experiment Videos
Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway
1Department of Medical Microbiology, Imperial College School of Medicine at St. Mary's, London, UK.
Abstract:
Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and beta-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.
Insights
Mycobacterium bovis bacille Calmette Guerin (BCG) survives in macrophages by residing in unique phagosomes, avoiding the host cell's lysosomal pathway. This study used organelle electrophoresis to identify these distinct compartments, crucial for understanding mycobacterial persistence.
Area of Science:
- Cell Biology
- Immunology
- Microbiology
Background:
- Mycobacteria are intracellular pathogens that persist within host phagocytes.
- Their survival is linked to modifying the intracellular environment, evading lysosomal degradation.
- The molecular mechanisms of mycobacterial entry and survival are not fully understood.
Purpose of the Study:
- To investigate the intracellular fate of Mycobacterium bovis bacille Calmette Guerin (BCG) within murine macrophages.
- To characterize the phagosomal compartments occupied by BCG using organelle electrophoresis.
- To identify host proteins associated with BCG-containing phagosomes.
Main Methods:
- Organelle electrophoresis to separate phagosomes from endosomal/lysosomal compartments.
- Biochemical analysis of purified mycobacterial phagosomes for endosomal/lysosomal markers (LAMP-1, beta-hexosaminidase).
- Subcellular fractionation combined with two-dimensional gel electrophoresis to analyze host proteins.
Main Results:
- Live BCG, but not dead BCG, resides in phagosomes physically separable from endosomal/lysosomal compartments.
- Purified BCG phagosomes lack common endosomal/lysosomal markers.
- A distinct set of host proteins is found in BCG phagosomes, absent in endosomal/lysosomal compartments.
Conclusions:
- Mycobacteria persist by residing in unique compartments outside the host cell's endosomal/lysosomal system.
- This sequestration may shield them from immune recognition.
- The organelle electrophoresis approach aids understanding of mycobacterial intracellular trafficking and host-pathogen interactions.