Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages

Abstract

Background: The interaction between macrophages and Cryptococcus neoformans (Cn) is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing 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 human immuno-deficiency virus (HIV) and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn.

Results: Live imaging of Cryptococcus 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.

Conclusion: 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 Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections.

Figure 1

Cell-to-cell transfer of C. neoformans leads to infection of previously uninfected cell. Following phagocytosis, macrophages closely apposed to each other underwent fusion leading to cell to cell transfer of Cn. The thin arrow indicates the Cn yeast that is being transferred while the wide arrow points to the Cn yeast that has been fully transferred to the previously uninfected murine macrophage cell. Images were collected at 20×.

Figure 2

Macrophage fusion and cell-to-cell Cn transfer involves actin. 3D reconstructed confocal images show infected macrophages fusing (A). Cutting along the Z-axis of panel (A) demonstrates that the intracellular Cn are being shared by both macrophages and are surrounded by a high concentration of phalloidin-labeled actin from both macrophages, as indicated by the arrow (B). Panel (C) shows an uninfected macrophage. Images were collected at 63×.

Figure 3

Massive vacuole formation follows Cn extrusion by macrophages. Macrophage extrusion of Cn cells was followed by the formation of a massive vacuole ranging up to approximately 15 μM in size. This formation was not observed in bead infected or uninfected macrophages. The thin arrow points to the nascent vacuole which progressively grows over time to a large vacuole, as indicated by the thick arrow. Images were collected at 10×.