Abstract
The Pacific Northwest outbreak of cryptococcosis, caused by a near-clonal lineage of the fungal pathogen Cryptococcus gattii, represents the most significant cluster of life-threatening fungal infections in otherwise healthy human hosts currently known. The outbreak lineage has a remarkable ability to grow rapidly within human white blood cells, using a unique ‘division of labour’ mechanism within the pathogen population, where some cells adopt a dormant behaviour to support the growth of neighbouring cells. Here we demonstrate that pathogenic ‘division of labour’ can be triggered over large cellular distances and is mediated through the release of extracellular vesicles by the fungus. Isolated vesicles released by virulent strains are taken up by infected host macrophages and trafficked to the phagosome, where they trigger the rapid intracellular growth of non-outbreak fungal cells that would otherwise be eliminated by the host. Thus, long distance pathogen-to-pathogen communication via extracellular vesicles represents a novel mechanism to control complex virulence phenotypes in Cryptococcus gattii and, potentially, other infectious species.
Highlights
The Pacific Northwest outbreak of cryptococcosis, caused by a near-clonal lineage of the fungal pathogen Cryptococcus gattii, represents the most significant cluster of life-threatening fungal infections in otherwise healthy human hosts currently known
We previously showed that outbreak strains of C. gattii induce the rapid intracellular proliferation of otherwise non-virulent strains during co-infection[6]
To test whether this effect required the fungal cells to be present within the same host cell, we generated fluorescently tagged versions of an outbreak (R2657) and nonoutbreak (ICB180) strain of C. gattii and confirmed that these strains were unaltered from their parental strains in morphology, growth or stress tolerance (Supplementary Fig. 1)
Summary
From R265 were significantly larger (26−397 nm, median size of 108 nm) than those from R265ΔCap[10] (median 72.8 nm) or ICB180 (median 86.3 nm), in agreement with data from C. neoformans showing reduced EV size in acapsular strains[14]. Addition of capsular material to the acapsular strain R265ΔCap[10] in vitro (Fig. 2b and Supplementary Fig. 2a) further suggested that the presence of an intact capsule secretion pathway is necessary to trigger raised IPRs in non-outbreak cryptococci. To test whether the EVs that are shed by C. gattii may be responsible for triggering the long-distance proliferation described above, we infected the non-outbreak strain ICB180 into macrophages and added 10 μg of EVs, isolated from the outbreak strain R265, to the media. EVs isolated from the virulent C. neoformans KN99 strain were unable to raise the IPR of ICB180 (Fig. 3e) Taken together, these data suggest that EVs act as an ‘accelerator’ of intracellular proliferation (and virulence) within C. gattii, but only for strains that already have the capacity for low (but not zero) rates of intracellular proliferation. We set out to establish which EV components might be responsible for triggering augmented IPRs
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