Real-time visualization of phagosomal pH manipulation by Cryptococcus neoformans in an immune signal-dependent way

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.

Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis

“…C . neoformans infection is known to cause phagolysosome membrane permeabilization [ 35 , 113 – 115 ], which potentially enables access to PA from the host cell cytosol. Moreover, metabolomic evidence in both human and mouse cells indicates that, especially under specific cellular stress conditions, lysosomes can serve as storage sites for amino acids and metabolites, including pantothenic acid, at concentrations reaching up to the micromolar range [ 116 , 117 ].…”

“…In bacteria, PA-auxotroph attenuates virulence in M. tuberculosis but not in Salmonella [111,112], reinforcing existing variations in the composition of the phagolysosome depending on the hosted pathogen. C. neoformans infection is known to cause phagolysosome membrane permeabilization [35,[113][114][115], which potentially enables access to PA from the host cell cytosol. Moreover, metabolomic evidence in both human and mouse cells indicates that, especially under specific cellular stress conditions, lysosomes can serve as storage sites for amino acids and metabolites, including pantothenic acid, at concentrations reaching up to the micromolar range [116,117].…”

Host–Pathogen Interactions and Correlated Factors That Are Affected in Replicative-Aged Cryptococcus neoformans

“…To analyze the pH alterations in the phagosomes infected with young and old C. neoformans, we used the amine-reactive pH dye Phrodo green AM (P35373, Thermo Fisher Scientific, Waltham, MA, USA) to label the surface of C. neoformans, according to the manufacturer's instructions and adapted from [15,25]. This staining is non-fluorescent at a neutral pH, but then becomes fluorescent at an acidic pH.…”

“…Based on pivotal studies that characterized C. neoformans interactions with macrophages [15], we hypothesized that C. neoformans phagosome interactions impose selection on the C. neoformans population in the host. In this study, we investigated the strategies used by old C. neoformans to thrive inside host macrophages, and we characterized the age-associated virulence factors that contribute to fungal intracellular survival.…”

Dendritic Cells: Multifunctional Roles in Host Defenses to Cryptococcus Infections

“…How C. neoformans affects the lysosomal maturation pathway is not well understood in DCs, but early studies in macrophages show C. neoformans does not prevent phagolysosome fusion and can survive in an acidic environment [79,80]. However, recent studies, mainly in monocytes and macrophages, have found that C. neoformans is capable of modulating the phagosome's pH and development [77,[81][82][83][84].…”