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Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm.

ABSTRACT: Cryptococcus neoformans (CN), an encapsulated, ubiquitous environmental yeast, is pathogenic for humans, primarily those with compromised immune function. CN is believed to be a facultative intracellular pathogen. Time-lapsed video microscopy revealed that yeast began to replicate and divide 2 hours after ingestion by J774.16 macrophage cells, with the average cell hosting 10-40 organisms of varying morphologies before ultimately lysing and releasing organisms, either singly or in clumps. Intracellular growth was accompanied by the accumulation of polysaccharide-filled vesicles in the macrophage. Studies with fluorescently labeled dextran revealed that the phagolysosomal compartment became leaky during the course of intracellular infection. Consistent with this observation, phagosomes containing CN had an increased pH relative to similar phagosomes containing inert magnetic beads, as indicated by a colorimetric change in the pH-sensitive Lysosensor dye. Immunocytochemistry revealed differences in the reactivity of polysaccharide elaborated by CN inside macrophages relative to that expressed in vitro. Taken together these results are suggestive of a novel mechanism of intracellular survival by an encapsulated organism, whereby ingestion is followed by damage to the phagosomal membrane resulting in continuity with the cytoplasm, accumulation of polysaccharide-containing vesicles, and possibly, production of a structurally different polysaccharide.

SUBMITTER: Tucker SC

PROVIDER: S-EPMC122490 | biostudies-literature | 2002 Mar

REPOSITORIES: biostudies-literature

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Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm.

Tucker Stephanie C SC Casadevall Arturo A

Proceedings of the National Academy of Sciences of the United States of America 20020301 5

Cryptococcus neoformans (CN), an encapsulated, ubiquitous environmental yeast, is pathogenic for humans, primarily those with compromised immune function. CN is believed to be a facultative intracellular pathogen. Time-lapsed video microscopy revealed that yeast began to replicate and divide 2 hours after ingestion by J774.16 macrophage cells, with the average cell hosting 10-40 organisms of varying morphologies before ultimately lysing and releasing organisms, either singly or in clumps. Intrac ...[more]

PMID: 11880650

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Project description:UnlabelledA unique aspect of the interaction of the fungus Cryptococcus neoformans with macrophages is the phenomenon of nonlytic exocytosis, also referred to as "vomocytosis" or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed only in vitro using subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major question still remaining is whether this phenomenon also occurs in vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosis in vitro and used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phagosomal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a significant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Although all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurred in vivo with a frequency that is possibly much higher than that observed in vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis.ImportanceCryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neoformans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis. To dissect this phenomenon, we evaluated its dependence on the pH inside the macrophage and addressed its occurrence during infection of mice. We developed new techniques using flow cytometry to measure C. neoformans internalization by and nonlytic exocytosis from macrophages. Neutralizing the phagosome acidity changed the rate of nonlytic exocytosis: activity increased with the weak bases chloroquine and ammonium chloride, whereas the vacuolar ATPase inhibitor bafilomycin A1 caused it to decrease. Experiments in mice suggested that nonlytic exocytosis occurred during infection with C. neoformans. These results shed new light on the interaction between C. neoformans and host macrophages.

Project description:Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.

Project description: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.

Dataset Information

Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm.

Publications

Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm.

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