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Phagosome-lysosome fusion in P388D1 macrophages infected with Histoplasma capsulatum
L G Eissenberg1, P H Schlesinger, W E Goldman
1Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.
Abstract:
The issue of whether or not phagocytized Histoplasma capsulatum yeasts evade phagosome-lysosome fusion (P-LF) has been debated by several investigators. To resolve this problem, yet avoid drawbacks associated with the conventional assays of P-LF (electron microscopy and the acridine orange assay), we used fluorescein isothiocyanate-labeled dextran (FITC-dextran) to monitor P-LF in the macrophage-like cell line P388D1.D2. Controls indicated that FITC-dextran could be used to distinguish between evasion of P-LF by Toxoplasma gondii and phagolysosome formation following ingestion of Saccharomyces cerevisiae. Phagosomes containing H. capsulatum clearly fused with FITC-dextran-labeled lysosomes at a rate comparable to that observed for S. cerevisiae. This was true for several strains of H. capsulatum including two avirulent strains derived in this laboratory. Varying the dose of H. capsulatum did not alter the percentage of phagolysosomes formed. Our results indicate that H. capsulatum is one of a small number of organisms which is able to survive in phagolysosomes.
Insights
Histoplasma capsulatum yeasts do not evade phagosome-lysosome fusion (P-LF). Instead, this fungal pathogen survives within the phagolysosome, challenging previous assumptions about its intracellular behavior.
Area of Science:
- Mycology
- Cell Biology
- Immunology
Background:
- The intracellular survival mechanisms of Histoplasma capsulatum are not fully understood.
- Previous studies have debated whether H. capsulatum yeasts evade phagosome-lysosome fusion (P-LF).
- Conventional assays for P-LF have limitations.
Purpose of the Study:
- To definitively resolve the debate on H. capsulatum's interaction with phagolysosomes.
- To establish a reliable method for monitoring P-LF in macrophage models.
- To investigate the survival strategy of H. capsulatum within host cells.
Main Methods:
- Utilized fluorescein isothiocyanate-labeled dextran (FITC-dextran) to track phagosome-lysosome fusion.
- Employed the macrophage-like cell line P388D1.D2 for experiments.
- Used Toxoplasma gondii and Saccharomyces cerevisiae as controls for assay validation.
Main Results:
- FITC-dextran effectively distinguished between phagolysosome evasion and formation.
- H. capsulatum phagosomes demonstrated fusion with FITC-dextran-labeled lysosomes.
- Fusion rates for H. capsulatum were comparable to those of S. cerevisiae across multiple strains.
- Phagolysosome formation was independent of H. capsulatum dose.
Conclusions:
- H. capsulatum yeasts do not evade phagosome-lysosome fusion.
- The pathogen survives within the phagolysosome, indicating a unique survival strategy.
- This finding identifies H. capsulatum among a select group of organisms capable of thriving in the phagolysosomal environment.