Article Figures & Data
Figures
- FIG 1
A. fumigatus induces EV release by human neutrophils. (A) Time course of apoptotic body (APB) occurrence (green lines) and fungus-induced cell death (teal lines) at MOIs of 5 and 10 (n = 10 [15] and n = 12 [17] for apoptotic body counts for MOIs of 5 and 10, respectively; n = 4 [20] and n = 5 [15] for viability data for MOIs of 5 and MOI 10, respectively) (numbers in brackets are total number of technical replicates). (B) Percentage of apoptotic bodies per total number of EVs. (C to E) Time course of total EV release and the levels of the EV surface markers annexin V (n = 27 [40] for sEVs, n = 16 for afEVs and pksP EVs) (C), CD11b (n = 23 for sEVs, n = 16 for afEVs and pksP EVs) (D), and CD63 (n = 13 for sEVs, n = 9 for afEVs and pksP EVs) (E). sEVs were collected from uninfected cells. Symbols represent significant differences between pksP EVs and afEVs (*), pksP EVs and sEVs (+), afEVs and sEVs (x). The data in panels A and B to E are presented as the medians and interquartile ranges of the absolute numbers of EVs per 107 PMNs. P values were determined by the Mann-Whitney test. *, P < 0.05; **, ++, and xx, P < 0.01; *** and xxx, P < 0.001. (F to M) Cryo-TEM images of sEVs (F to I) and afEVs (J to M) at 2 h postinteraction. Representative images display sEVs (G to I) and afEVs (K to M) with different appearances. Bars, 200 nm.
- FIG 2
Analysis of the EV proteome by LC-MS/MS reveals that neutrophil-derived EVs retain a core proteome cargo after infection. (A) Venn diagram (created with Venny [version 2.1.0] software) indicating the overlap of proteins identified from each EV population using label-free quantification. (B to D) Volcano plots comparing proteins identified in afEVs, pksP EVs, and sEVs using the LFQ-based proteomics method.
- FIG 3
afEVs elicit antifungal effects on wt fungus. (A) Representative bright-field images after 10 h of incubation of wt fungal hyphae with afEVs and pksP EVs. Single (1×) or triple (3×) doses of EVs were applied. (B to E) Growth of wt fungal hyphae after 10 h of coincubation with afEVs and pksP EVs derived from four different donors. The size of the hyphae was assessed by automated analysis of 2D image data, and the results are displayed as the median hyphal area in each field of view; data are represented as medians and interquartile range of the median hyphal area in each field of view (n = 10 fields of view per condition per time point). (F) Representative growth curves of the wt fungal strain in the presence and the absence of EVs for the donor for which the results are shown in panel D. (G) Effects of sEVs on wt conidia compared to those of afEVs on wt conidia (n = 3 independent experiments, 20 fields of view per experiment per condition). P values were determined by the Mann-Whitney test. n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.
- FIG 4
Effect of afEVs on hyphae. (A) Density of afEVs and pksP EVs inside and outside of wt and pksP mutant hyphae. (B) The fraction of PI-stained hyphae indicates permeable fungal hyphae and provides an estimation of the hypha-associated DNA signals in wt and pksP hyphae treated with afEVs and pksP EVs, respectively, compared to those in untreated control hyphae. The data in panels A and B for the EV groups were derived from 3 independent experiments (n = 13 and 21 technical replicates for pksP and wt, respectively). The data in panel B for the controls are representative of those from 1 experiment (n = 5 technical replicates). P values were determined by the Mann-Whitney test. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) SEM images of 50-h-old cultures of wt hyphae treated with afEVs (bottom) versus healthy hyphae grown alone (top). Samples were immobilized on filter membranes with a defined pore size of 5 μm (black circles). Bars, 50 μm. SEM images represent observations from 2 independent experiments with 3 technical replicates.
- FIG 5
afEVs kill fungal hyphae. AfS35/pJW103 hyphae expressing a mitochondrial GFP reporter (green) grown for 20 h were stained with calcofluor white (blue) and incubated with sEVs, afEVs, pksP EVs, or 3 mM H2O2 as a positive control for cell death induction or left untreated and then monitored by CLSM. A healthy filamentous mitochondrial network is shown in green in an untreated sample. A fragmented mitochondrial network indicates cell death, as seen when 3 mM H2O2 was used as a positive control for cell death. Images are representative of those from 4 separate experiments with samples from different donors. Bars, 10 μm.
- FIG 6
afEVs are internalized into the fungal cell wall and cytoplasm. afEV internalization into fungi was analyzed by 3D quantitative analysis of z-stacks with GFP-expressing A. fumigatus after 20 h of coincubation. (A, B) (Left) Representative cross sections of z-stacks showing lateral (X and Y) and axial (Z) dimensions of a hypha with internalized afEVs (A) and the corresponding control hypha (B). Internalized afEVs are in red (Alexa Fluor 647), the fungal cell wall is in blue (calcofluor white), and the fungal cytoplasm is in green (GFP). The image intensity was inverted. The darkest color corresponds to the highest fluorescence intensity. Bars, 2 μm. (Right) Histograms display the specificity of the signal of the Alexa Fluor 647 dye used to stain afEVs. As seen in the control z-stack, there is unspecific Alexa Fluor 647 staining, likely due to dye aggregation. (C) Schematic diagram of a cross section of hyphae and different stages of afEV internalization. afEVs were located in 4 areas, as indicated by the graphical representation. (D) Overview from the 3D image analysis of different locations of afEVs. Data are representative of those from 3 independent experiments with a total of 25 z-stacks.
- FIG 7
The intracellular production of human azurocidin and cathepsin G proteins is toxic to A. fumigatus. (A) A. fumigatus wt and mutant strains Afazuro, AfcathG, and AfRBP7 harboring the human azurocidin, cathepsin G, and RBP7 genes, respectively, under the control of the tetON promoter. The cultures were grown for 24 h in the absence or the presence of doxycycline (DOX). (B) Biomass measurement of wt and A. fumigatus mutant strains Afazuro, AfcathG, and AfRBP7 with and without doxycycline. Data are representative of those from 3 independent experiments with 3 technical replicates. P values were determined by the Mann-Whitney test. ns, not significant; **, P < 0.01; ***, P < 0.001. (C) Detection of proteins produced in the A. fumigatus mutant strains. The bar plot shows the abundance level of the azurocidin protein for the Afazuro strain, the cathepsin G protein for the AfcathG strain, and RBP7 for the AfRBP7 strain, based on the intensity of the precursor ion. The data were generated from 3 analytical replicates.
Tables
- TABLE 1
Selected examples of differentially produced proteins with known antimicrobial activity
FIG S1
Flow cytometry analysis of PMN-derived EVs. (A) Purity of human PMNs used to generate PMN-derived EVs. Purity was assessed by flow cytometry based on staining for CD66b+ CD16b+ PMNs, CCR3+ eosinophils, CD14+ monocytes, CD3+ T cells, CD19+ B cells, CD56+ NK cells, and NKT cells. The predominant contaminating fraction of cells was eosinophils. (B to C) Analysis of apoptotic bodies defined as double-positive (annexin V- and PI-positive) EVs. PMNs cultured for 3 days on Dulbecco modified Eagle medium at 37°C were used as a positive control for apoptosis (left). The results for two donors are shown in the center and right panels. (D and E) Flow cytometry protocol for phenotyping afEVs. (D) The instrumental background noise by pure HBSS was recorded and subsequently subtracted from the values for all samples. (E) The results for midintensity rainbow beads of 3.8 μm were recorded to set the upper detection limit; afEVs are detected in the gate above the noise and below the beads. (F and G) Single-stained afEVs with the corresponding isotype antibodies were used as negative controls. Stained afEV suspensions were measured before (F) and after (G) detergent treatment with 1% (vol/vol) Triton X-100 to verify the vesicular nature of the detected events. False-positive events (detergent resistant) were subtracted from the results. Download FIG S1, TIF file, 0.1 MB.
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FIG S2
Characterization of afEV surface markers by flow cytometry. (A) Flow cytometry measurement of PMN surface marker dynamics of CD11b and CD63 during infection with wt and pksP conidia at an MOI of 5. PMNs were gated according to forward scatter/side scatter properties, dead cells were excluded by staining with viability Zombie dye, and the expression of CD11b and CD63 was analyzed with FlowJo software (TreeStar). (B) Size distribution of afEVs, pksP EVs, and sEVs generated at different time points, as measured by dynamic light scattering. Data are representative of those from 3 independent experiments. (C) Time course of apoptotic body occurrence (green lines) compared to that of fungus-induced cell death (teal lines) for wt and pksP infected PMNs. Data are represented as the medians and interquartile ranges. Data for EVs are shown as absolute or relative vesicle numbers per 107 PMNs. P values were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Opsonization of wt and pksP mutant conidia as determined by flow cytometry for C3 immunofluorescence staining. Bars indicate the mean fluorescence intensity plus standard deviation from 2 experiments with 5 replicates each. Download FIG S2, TIF file, 0.1 MB.
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FIG S3
Neutrophil EV composition differs depending on the stimuli. (A to C) Volcano plots comparing proteins identified in afEVs, pksP EVs, and sEVs using the TMT-labeling proteomics method. (D) Gene Ontology (GO)-term enrichment analysis of the core proteome cargo (60 proteins), based on the FungiFun2 tool, reveals the pathways of EV biogenesis. The data are representative of those from 2 technical replicates. Download FIG S3, TIF file, 0.3 MB.
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TABLE S1
Identified proteins with transmembrane domains predicted by use of the SignalP, TMHMM, and WoLF PSORT tools based on the TMT and LFQ data sets obtained here. Download Table S1, PDF file, 0.1 MB.
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FIG S4
Effect of afEVs on pksP mutant fungal cells. (A) Segmentation steps of an automated algorithm for 2D image analysis of fungal growth with (top rows) and without (bottom rows) afEVs. Bars, 20 μm. (B) Representative bright-field images after 10 h of incubation of fungi with afEVs and pksP EVs on pksP mutant hyphae. Untreated hyphae received no EVs. Single (1×) or triple (3×) doses of EVs were applied as described in Materials and Methods. (C to F) Growth of pksP mutant fungal hyphae after 10 h of coincubation with afEVs and pksP EVs derived from four different donors. The size of the hyphae was assessed by automated analysis of 2D image data, and the results are displayed as the median hyphal area (in square micrometers) in each field of view; data are represented as the medians and interquartile ranges of the median hyphal area in each field of view (n = 10 fields of view per condition per time point). (G) Representative growth curves of the pksP mutant fungal strain in the presence and absence of EVs for the donor for which the results are shown in panel E. Download FIG S4, TIF file, 0.2 MB.
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FIG S5
Testing of hyphae and EV volume. (A, B) Equal volumes of EVs (A) and hyphae (B) for wt and pskP samples were analyzed. (C) Dependence of the volume fraction of the hypha-associated afEVs (volume of hypha-associated afEVs divided by the total afEV volume) on the volume concentration of afEVs (total afEV volume divided by the sample volume). (D) Dependence of the volume fraction of hypha-associated EVs on the volume concentration of hyphae (hyphal volume divided by the sample volume). (E) Dependence of the volume fraction of the hypha-associated DNA (HADNA) that interacts with EVs on the volume fraction of hypha-associated EVs. Download FIG S5, TIF file, 0.1 MB.
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FIG S6
SEM imaging of afEV-treated hyphae. SEM images of 50-h-old cultures of wt hyphae treated with afEVs (bottom) versus healthy hyphae grown alone (top). Samples were immobilized on filter membranes with a defined pore size of 5 μm (as seen in the overview images). Bars, 1 μm (images on the far right) and 5 μm (all other images). SEM images represent observations from 3 technical replicates from 2 independent experiments. Download FIG S6, TIF file, 0.2 MB.
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FIG S7
Verification of A. fumigatus transgenic strains expressing human EV proteins. (A) Schematic of the predicted BamHI restriction sites in the genomes of the mutant strains following integration of the constructs used to express the human genes in the parental strain: the tetON promoter, the azurocidin-encoding gene, the cathepsin G-encoding gene, the RBP7-encoding gene, the tef terminator, and the ptrA cassette (pyrithiamine resistance marker). (B) Southern blot for confirmation of construct integration into the A. fumigatus genome. In the transgenic strains, bands with the expected size of 1.7 kb for the Afazuro strain, 1.4 kb for the AfcathG strain, and 1.4 kb for the AfRBP7 strain were observed. No signal was detected for the nontransformed wt strain. Download FIG S7, TIF file, 0.1 MB.
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TABLE S2
Primers used in this study. Download Table S2, PDF file, 0.01 MB.
Copyright © 2020 Shopova et al.
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