Rethinking mechanisms of intracellular destruction of pathogens

Abstract

Cryptococcus neoformans is an encapsulated fungus that develops predominantly within immunocompromised hosts. Recent studies have shed light on the molecular and cellular basis of its pathogenesis. For example, the capsular layer of C. neoformans is required for virulence; it also elicits an antiphagocytic effect in vitro and shedding of the capsule promotes immunosuppression in vivo. C. neoformans liberates parts of its capsule after ingestion, which leads to the development of a large intracellular phagosome compartment. Dissecting this unique mechanism of pathogenesis in vivo has been difficult. As a result, in vitro models have been the primary means of examining C. neoformans invasion of host cells.A recent study by Tucker and Casadevall [1xReplication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Tucker, S.C. and Casadevall, A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3165–3170Crossref | PubMed | Scopus (160)See all References][1] provides strong evidence that C. neoformans replicates in the cytoplasm of macrophages using a mechanism involved in increasing phagosome permeability, with concomitant formation of intracellular vesicles that contain capsular polysaccharide. Intracellular shedding of capsular substances was measured using an immunofluorescence detection approach coupled with phase imaging. Small vesicles in the cytoplasm that stained with anti-capsule antiserum could be seen and were distinct from the larger, more brightly stained intracellular fungal cells. The authors examined loss of phagosomal membrane integrity using transmission electron microscopy, which revealed discontinuity between the phagosomal membrane and fungal cells in compartments containing engulfed C. neoformans. To determine whether membrane dissolution in phagosomes could be a mechanism by which C. neoformans could sequester nutrients, they measured the permeability of preloaded intracellular vesicles containing fluorescent markers in both infected and uninfected macrophages. Phase images of cells infected with C. neoformans captured the movement and diffusion of the fluorescent markers after fusion with the phagosome, indicating that there was increased permeability of the phagosomal membrane. To substantiate this observation, pH measurements using lysosensor staining of acidic compartments revealed that phagocytic vesicles containing fungal cells were less acidic than uninfected control macrophages, suggesting a loss of phagosome membrane integrity.Collectively, these data demonstrate that phagocytic uptake of C. neoformans by macrophages subsequently leads to dissolution of the phagosomal compartment, enabling the fungus to replicate intracellularly. In addition, there is simultaneous accumulation of polysaccharide in cytoplasmic vesicles. The infection process ultimately culminates in the lysis and destruction of the host cell. These data present a microbial strategy of subverting intracellular destruction that is different to the mechanisms used by other well-studied intracellular pathogens. For example, Legionella pneumophila prevents lysosome–phagosome fusion, and Listeria monocytogenes uses vacuole acidification for enzyme activation and subsequent lysis of the phagosomal compartment. In C. neoformans pathogenesis, the role of these intracellular polysaccharide vesicles and permeabilizing phagosomal membranes might provide more insight into this interesting mechanism.