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Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages
S M Levitz1, S H Nong, K F Seetoo
1Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, Massachusetts, USA.slevitz@bu.edu
Abstract:
Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.
Insights
Human macrophages (MDM) fight Cryptococcus neoformans better when their phagolysosomes are less acidic. This study shows C. neoformans thrives in acidic environments within macrophages.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Human monocyte-derived macrophages (MDM) exhibit enhanced antifungal activity against Cryptococcus neoformans when treated with alkalinizing agents.
- This suggests that the phagolysosomal pH plays a critical role in controlling C. neoformans growth.
- Cryptococcus neoformans demonstrates an inverse correlation between growth and pH in cell-free media, indicating adaptation to acidic conditions.
Purpose of the Study:
- To determine the pH of phagosomes containing C. neoformans within human MDM and neutrophils.
- To investigate whether C. neoformans actively regulates phagosomal pH.
- To confirm phagosome-lysosome fusion in MDM infected with C. neoformans.
Main Methods:
- Fungi were labeled with pH-sensitive probes (fluorescein and Oregon Green) for phagosomal pH measurement.
- Phagosomal pH was assessed in MDM and neutrophils after C. neoformans ingestion.
- Chloroquine was added to assess its effect on phagosomal pH.
- Immunofluorescence microscopy was used to detect colocalization of C. neoformans with LAMP-1.
Main Results:
- The average phagosomal pH was approximately 5 and remained constant, indicating C. neoformans does not actively regulate phagosomal pH.
- Chloroquine treatment increased phagosomal pH significantly (up to 7.3 at 100 microM).
- C. neoformans-containing phagosomes colocalized with LAMP-1, confirming fusion with lysosomal compartments.
Conclusions:
- Cryptococcus neoformans resides and survives in an acidic phagolysosome within human macrophages.
- Unlike many intracellular pathogens, C. neoformans does not evade phagosome-lysosome fusion.
- The acidic phagolysosomal environment is crucial for C. neoformans survival within macrophages.