Cell-to-cell spread of intracellular pathogens is known to occur with viruses and bacteria such as HIV and Shigella
spp., respectively [10
]. Among the fungi, cell-to-cell spread could potentially occur when ingested cells form hyphae that protrude into neighboring cells, in a phenomenon that is associated with damage to both the donor and the host cell, although it is uncertain whether this has been described experimentally. Furthermore, we are not aware of any previous reports of cell-to-cell spread of yeast cells. Cell-to-cell spread is an important cellular pathogenic strategy because it allows cellular infection directly from the intracellular space and thus avoids exposure of the microbe to antimicrobial compounds in the extracellular space such as specific antibody and complement. Cell-to-cell spread can also protect microbes against certain antimicrobial drugs that have limited penetration into cells.
Cell-to-cell spread of C. neoformans was a relatively rare event. The phenomenon was observed with murine macrophages and macrophage-like cells under in vitro conditions, thus caution is warranted when extrapolating the relevance and importance of this phenomenon to in vivo situations. Nevertheless, the fact that it was observed provides the important precedent that cell-to-cell spread of yeast cells can occur. In this regard we note that in viral and bacterial systems cell-to-cell spread has also been documented only in vitro, yet is assumed to be possible in vivo.
cell-to-cell spread required contact between macrophage cells. The likely involvement of actin in this process was inferred by the observation that cell-to-cell transfer required the accepting cell to make contact with the donor cell through cytoplasmic projections that are abolished by the actin inhibitor cytochalasin D, in addition to confocal images shown in the results demonstrating actin rich regions from both fusing macrophages surrounding the cryptococcal cell. Although the mechanism for cell-to-cell transfer is unknown we note that cell membranes of infected cells appeared to be modified as evidenced by the extrusion of cryptococcal vacuoles with survival of the host cell and occasional cell-to-cell fusion events [5
]. Hence, cell-to-cell transfer of C. neoformans
may require microbial modification of the donor cell membrane followed by contact with an uninfected cell to create cell-to-cell interactions propitious for yeast transfer. This novel observation of Cn transfer from cell-to-cell raises the possibility that a similar mechanism could promote the dissemination of Cn during infection. For example, it is conceivable that cell-to-cell spread contributes to Cn dissemination in the CNS after crossing the blood-brain barrier. In addition to cell-to-cell spread, we report the formation of massive vacuole structures in macrophage-like cells that had previously harbored cryptococcal cells which had been released by the phenomenon of phagosome extrusion. Massive vacuole formation suggests that Cn intracellular habitation and replication in macrophage-like cells is associated with cellular damage, despite the fact that macrophages still remain alive as evidenced by their movement '[see Additional file 2
]'. Perhaps longer imaging periods may have been needed to witness any cell death resulting from this event. The mechanism of massive vacuole formation is not known but we surmise that it may arise from the fusion of empty phagosomal remnants and perhaps polysaccharide containing vacuoles. In this regard, Cn phagosomal extrusion is associated with the retention of polysaccharide-containing vesicles that may then fuse with themselves or promote the fusion of other cellular membranes. Irrespective of the mechanism involved in massive vacuole formation, the presence of large vacuoles in previously infected cells provides evidence for the cytotoxicity of Cn intracellular residence. The occurrence of this event during infection could contribute to local tissue pathogenesis and promote Cn survival and persistence of infection.