Novel interactions between Cryptococcus neoformans and macrophages: Intracellular persistence of the yeast leads to phagosomal extrusion, host cell-to-cell spread, and micro-colony formation following extrusion

Abstract

Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease in immunocompromised subjects. The interaction of C. neoformans with alveolar macrophages is critical for containing the infection but C. neoformans can also replicate intracellularly and lyse macrophages.;We initiated our studies by closely examining the C. neoformans and host macrophage interaction and found that under certain conditions, including phagosomal maturation, possible actin depolymerization and homotypic phagosome fusion, C. neoformans can exit the macrophage host through the extrusion of the phagosome, while both the released pathogen and host macrophage remain alive and able to propagate. The phenomenon of 'phagosomal extrusion' indicates the existence of a previously unrecognized mechanism whereby a fungal pathogen can escape the intracellular confines of a mammalian host to continue propagation and possibly, to disseminate.;The interaction between macrophages and C. neoformans is critical for containing dissemination of this pathogenic yeast. As previously mentioned, C. neoformans can either lyse macrophages or escape from within them through phagosomal extrusion. Both events result in live extracellular yeasts capable of replicating and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the bacteria Listeria monocytogenes and macrophages. In this report we have used time-lapse imaging to determine if this event occurs with C. neoformans. Live imaging of C. neoformans' interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare, its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that Cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells. This demonstrates that C. neoformans can escape the intracellular confines of macrophages in an actin-dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized C. neoformans cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage.;In another study, we explored the consequences of antibody-mediated phagocytosis for two closely related human pathogenic fungal species, C. neoformans and C. gattii, of which C. neoformans encompasses two varieties: neoformans and grubii. The interaction between C. neoformans varieties grubil and neoformans and host cells has been extensively studied, but that of C. gattii and macrophages remains largely unexplored. Opsonization of C. gattii cells with IgM mAb induced a change in the capsule structure, exposing the CD18 binding sites and promoting ingestion by macrophages through the complement receptor in the absence of complement components. In addition, like C. neoformans, antibody-mediated phagocytosis of C. gattii cells was followed by intracellular replication, host cell cytoplasmic polysaccharide accumulation and phagosomal extrusion. Further, both C. gattii and C. neoformans cells were extruded from and exited macrophages in biofilm-like microcolonies where the yeast cells were aggregated in a polysaccharide matrix that contained bound antibody. In contrast, C. neoformans variety grubii cells that were opsonized with complement were extruded from macrophages as dispersed individual cells. Thus, both antibody- and complement-mediated phagocytosis resulted in intracellular replication but the mode of opsonization affected the outcome of extrusion. The phenomenom of biofilm-like microcolony exit strategy of C. neoformans and C. gattii following antibody opsonization appeared to reduce fungal cell dispersion, suggesting that antibody agglutination effects persist in the phagosome to entangle nascent daughter cells, and contribute to antibody-mediated protection. (Abstract shortened by UMI.).