| Summary: | The pathogenic fungi Cryptococcus neoformans and Cryptococcus gattii are two of the main causes of life-threatening meningoencephalitis in immunocompromised and immunocompetent individuals respectively. Following inhalation, cryptococci are engulfed by phagocytic cells in the lung and previous studies by our group and others have demonstrated that they are then able to survive inside these cells (especially macrophages), thus acting as intracellular parasites. This intracellular phase is thought to underlie the ability of the pathogens to remain latent for long periods of time within infected individuals. Here, we demonstrate that cryptococci can also manipulate host macrophages in order to mediate an exquisitely controlled ‘escape’ process. This expulsive process, which we have termed ‘vomocytosis’, can occur either into the extracellular milieu or, remarkably, into neighbouring host cells, thus resulting in direct cell-to-cell transmission (‘lateral transfer’). After vomocytosis, both the host macrophages and the expelled cryptococci appear morphologically normal and continue to proliferate. Vomocytosis therefore represents an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation. Moreover, direct cell-to-cell spread of cryptococci allows the pathogen to remain concealed from the immune system and protected from antifungal agents, thus achieving long-term latency. This project has also provided a possible explanation for the molecular cause of a recent C. gattii outbreak on Vancouver Island, Canada. We found that isolates from the outbreak have dramatically increased their ability to replicate within macrophages in comparison with other C. gattii strains, despite the fact that they are genetically very similar to each other. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this ability might be the cause of the outbreak. Finally, application of high-density whole genome tiled arrays, confocal microscopy and mating assays reveal regulation of mitochondrial activity to be a major driver of virulence in this pathogen group. Taken together, these data indicate that a recent change in mitochondrial regulation within the C. gattii lineage has led to an increased intracellular proliferative capacity, resulting in the hypervirulent phenotype that underlies the outbreak. Such shifts in intracellular replication capacity may be a widespread phenomenon in other human pathogens and could potentially underpin disease epidemics caused by otherwise unrelated pathogens.
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