Fig. 6 | Nature Communications

Fig. 6

From: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii

Fig. 6

EVs added to macrophages infected with acapsular R265-GFP rapidly colocalise with the cryptococcal phagosome. a Macrophages alone (MO; i), macrophages with phagocytosed R265ΔCap10-GFP cells (ii) or macrophages containing R265ΔCap10-GFP and co-incubated with EVs isolated from R265ΔCap10 (EVsR265ΔCap10; (iii)) are not recognised by capsule-specific MAb 18B7. Those macrophages co-incubated with EVs isolated from R265 (EVsR265; (iv)), with capsule isolated from R265 (CapsuleR265; (v)) or heat-inactivated EVs isolated from R265 (EVsR265hk; (vi)) show colocalisation between R265ΔCap10-GFP cells and red signal suggesting that the EVs and capsule polysaccharides were delivered to the phagosomes. Pictures represent maximum intensity projections of 21 z-stacks obtained from 10 μm cross section through macrophages. Bars: 10 and 2 μm. b Graph showing percentage of colocalised signals of GFP labelled yeast cells R265ΔCap10 engulfed by murine macrophages and MAb 18B7-immunostained capsule (+capsule), heat-inactivated EVs isolated from R265 (EVsR265hk) or EVs isolated from R265 (+EVsR265). As a negative control intensities from macrophages infected with R265ΔCap10-GFP without EVs were used (+none). All numbers are given as means ± standard deviation (s.d.) and are representative of results from 3 to 4 independent experiments with a minimum of 25 macrophages analysed per sample per experiment. ns indicate statistically non-significant difference (Kruskal−Wallis test, P = 0.89) and quadruple asterisks indicate a highly significant difference (****, unpaired Mann−Whitney test, P 0.0001)

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