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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
Published on: December 7, 2015
Real-time visualization of phagosomal pH manipulation by Cryptococcus neoformans in an immune signal-dependent way
Emmanuel J Santiago-Burgos1, Peter V Stuckey1, Felipe H Santiago-Tirado1,2,3
1Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, United States.
Abstract:
Understanding of how intracellular pathogens survive in their host cells is important to improve management of their diseases. This has been fruitful for intracellular bacteria, but it is an understudied area in fungal pathogens. Here we start elucidating and characterizing the strategies used by one of the commonest fungal pathogens, Cryptococcus neoformans, to survive intracellularly. The ability of the fungus to survive inside host cells is one of the main drivers of disease progression, yet it is unclear whether C. neoformans resides in a fully acidified, partially acidic, or neutral phagosome. Using a dye that only fluoresce under acidic conditions to stain C. neoformans, a hypha-defective Candida albicans mutant, and the nonpathogenic Saccharomyces cerevisiae, we characterized the fungal behaviors in infected macrophages by live microscopy. The main behavior in the C. albicans mutant strain and S. cerevisiae-phagosomes was rapid acidification after internalization, which remained for the duration of the imaging. In contrast, a significant number of C. neoformans-phagosomes exhibited alternative behaviors distinct from the normal phagosomal maturation: some phagosomes acidified with subsequent loss of acidification, and other phagosomes never acidified. Moreover, the frequency of these behaviors was affected by the immune status of the host cell. We applied the same technique to a flow cytometry analysis and found that a substantial percentage of C. neoformans-phagosomes showed impaired acidification, whereas almost 100% of the S. cerevisiae-phagosomes acidify. Lastly, using a membrane-damage reporter, we show phagosome permeabilization correlates with acidification alterations, but it is not the only strategy that C. neoformans uses to manipulate phagosomal acidification. The different behaviors described here provide an explanation to the confounding literature regarding cryptococcal-phagosome acidification and the methods can be applied to study other intracellular fungal pathogens.
Insights
Cryptococcus neoformans manipulates phagosome acidification within host cells, unlike other fungi. This study reveals varied acidification behaviors, impacting disease progression and offering insights into fungal pathogen survival strategies.
Area of Science:
- Cell Biology
- Mycology
- Immunology
Background:
- Intracellular pathogen survival mechanisms are crucial for disease management, yet fungal pathogens are understudied compared to bacteria.
- Cryptococcus neoformans is a common fungal pathogen, and its intracellular survival strategies, particularly within host phagosomes, are not well understood.
- The acidic environment of phagosomes is critical for pathogen clearance, but C. neoformans' interaction with phagosomal pH remains unclear.
Purpose of the Study:
- To elucidate and characterize the strategies employed by Cryptococcus neoformans for intracellular survival within host cells.
- To investigate the phagosomal maturation and acidification dynamics of C. neoformans during macrophage infection.
- To compare the intracellular behavior of C. neoformans with other fungal species like Candida albicans and Saccharomyces cerevisiae.
Main Methods:
- Utilized a pH-sensitive dye to monitor phagosomal acidification in macrophages infected with C. neoformans, a Candida albicans mutant, and Saccharomyces cerevisiae via live microscopy.
- Employed flow cytometry to quantify phagosomal acidification in a larger population of infected cells.
- Assessed phagosome integrity using a membrane-damage reporter to correlate with observed acidification patterns.
Main Results:
- Significant populations of C. neoformans-containing phagosomes displayed aberrant acidification: some acidified then lost acidity, while others never acidified.
- In contrast, phagosomes containing Candida albicans mutant and Saccharomyces cerevisiae rapidly acidified and maintained low pH.
- Phagosome permeabilization was linked to altered acidification, but C. neoformans employs additional strategies to manipulate phagosomal pH.
Conclusions:
- Cryptococcus neoformans actively manipulates phagosomal acidification, exhibiting diverse behaviors not seen in other fungi, which explains conflicting literature.
- These varied intracellular survival strategies are influenced by host cell immune status.
- The methodologies developed can be applied to study the intracellular survival mechanisms of other fungal pathogens.
