Fig 1.
Physical characterization of E. histolytica EVs.
(A) Workflow used for the isolation of EVs from E. histolytica-conditioned medium. Differential centrifugation of supernatants was performed at increasing speeds for 15 min each to clear the samples of cellular debris. EVs were pelleted by ultracentrifugation at 100,000 g for 1 h and washed once with filtered PBS prior to further use. Created in BioRender. (B, C) Nanoparticle tracking measurements of EhA1 EVs (B) and EhB2 EVs (C) (data averaged from 5 videos per sample, overlay of multiple independent measurements, n = 12–13). (D) Comparison of the modal particle size of EhA1 EVs and EhB2 EVs as determined by nanoparticle tracking (n = 9–11, unpaired t test, ns = not significant). (E) Comparison of the particle concentration of EhA1 and EhB2 EV samples in a set of standardized experiments (n = 3-4, unpaired t test, ns = not significant). (F) Transmission electron microscopic visualization of EhEVs subjected to immunogold labeling with rabbit anti-galactose/N-acetylgalactosamine (Gal/GalNAc) lectin or mouse anti-lipopeptidophosphoglycan (LPPG) primary and gold-conjugated secondary antibodies. Arrowheads indicate sites of labeling on individual EVs. Shown are representative images of one sample for EhA1 and EhB2 each.
Fig 2.
Comparison of the E. histolytica vesicle and trophozoite proteomes.
(A, B) Quantitative comparison of EhA1 and EhB2 EV (A) and trophozoite (B) proteomes. Shown are proteins significantly differentially abundant between two proteomes (FDR p < 0.05, s0 = 0.5, fold change ≥ |2|) (yellow) as well as proteins present in only one of both proteomes (blue/ green). Proteins were considered to be a part of the respective proteome if they were present in at least 2/3 samples. Hence, we differentiated between proteins completely unique to one proteome (absent from all samples of the other proteome) and proteins belonging to only one proteome but not unique (present in 1/3 samples of the other proteome). (C) Overview of the total number of proteins, hypothetical proteins, proteins with a transmembrane (TM) domain or signal peptide in each of the analyzed proteomes based on annotations in AmoebaDB release 68 [23]. (D) Schematic overview of the E. histolytica EV proteome with selected proteins of interest. Created in BioRender. (E) Venn diagram depicting the number of proteins differentially abundant in EhA1 compared with EhB2 EVs, as well as proteins differentially abundant in EhA1 compared with EhB2 trophozoites and amount of proteins common to both datasets. (F) Selected molecular function GO terms associated with proteins enriched or depleted in EV proteomes compared with trophozoite proteomes, based on statistical overrepresentation test performed with Panther knowledgebase [27,28].
Fig 3.
Analysis of novel miRNAs in E. histolytica EVs.
(A) Comparison of the number of miRNAs detected in EhA1 compared with EhB2 EVs, based on de novo miRNA prediction using BrumiR algorithm version 3.0 [31] (n = 3 for each clone). (B–E) Quantitative analysis of potential targets of novel E. histolytica miRNAs in the Homo sapiens and E. histolytica genome, predicted using miRanda algorithm version 3.3a [33,34]. Shown are the amount of potential targets per miRNA in the H. sapiens (B) and E. histolytica (C) genome, as well as the amount of miRNAs potentially binding to the same target (D and E). (F) Normalized expression values of the two miRNAs detected only in EhA1 and not EhB2 EVs (EDGE test, *** FDR p < 0.001, n = 3). (G) Molecular function GO terms associated with identified miR-200 targets in the human genome (analysis performed with shinyGO version 80 [37], shown are the top 20 GO terms). (H) Amount of annotated versus hypothetical or unspecified predicted targets of miR-200 in the E. histolytica genome (according to AmoebaDB version 68 [23]). (I) Molecular function GO terms associated with miR-200 targets in the E. histolytica genome with an annotated function (analysis performed with shinyGO version 80).
Fig 4.
Cellular uptake of E. histolytica EVs and activation of primary monocytes.
(A) Graphical depiction of the workflow used for the determination of EV uptake by primary monocytes. Bone-marrow derived monocytes from male (shown as dots in graphs) and female (shown as triangles) mice were stimulated for 30 min with 0.5 µg EVs labeled with BCECF or corresponding volumes of controls and cellular uptake was quantified by spectral flow cytometry. Created in BioRender. (B) Dot plots of representative samples depicting flow cytometric identification of BCECF-positive cells following stimulation with mock control, EhA1 EVs or EhB2 EVs labeled with BCECF. (C) Percent BCECF+ monocytes after stimulation based on gates in (B). (D) Median fluorescence intensity (MFI) of BCECF in single cells after stimulation. MFIs were normalized to monocytes stimulated with unlabeled EVs for each independent experiment. (Kruskal-Wallis test with Dunn‘s multiple comparisons test, *p < 0.05, **p < 0.01, n = 6 from 3 independent experiments). (E) Dot plots of representative samples depicting surface expression of the activation marker CD38 on Ly6Chi monocytes upon stimulation. Monocytes were stimulated in vitro for 24 h with 1000 EVs/cell of EhA1 or EhB2 EVs or corresponding volume mock control and subsequently analyzed by flow cytometry. (F) Percent CD38+ Ly6Chi monocytes following EV stimulation. To control for the effect of protein denaturation on stimulatory capacity, EV and control samples were heat inactivated (h.i.) at 95°C for 10 min prior to stimulation. (G) Percent CD38+ Ly6Clo monocytes following EV stimulation. (One-way ANOVA with Šídák‘s multiple comparisons test, *p < 0.05, **** p < 0.0001, n = 6).
Fig 5.
Transcriptional profile of EV-stimulated monocytes.
Male and female bone marrow-derived monocytes were stimulated for 8 h in vitro with 1000 EVs/cell or equal volume mock control. mRNA expression levels were subsequently analyzed using RNA sequencing. (A) Heatmap depicting expression levels of genes significantly differentially expressed between EhA1 EV and/or EhB2 EV-stimulated monocytes and mock controls (fold change ≥ |2|, Bonferroni-corrected p < 0.05, n = 4). (B, C) Volcano plots depicting relative gene expression between EhA1 EVs (B) or EhB2 EVs (C) and mock controls. Genes significantly upregulated in EV-stimulated monocytes are depicted in red, whereas genes significantly downregulated are depicted in blue (log fold change ≥ |1|, Bonferroni-corrected p < 0.05, n = 4). Selected genes of interest are labeled. (D, E) Molecular function GO term analysis of significantly upregulated genes in EhA1 EV (D) or EhB2 EV (E)-stimulated monocytes compared with mock controls (analysis performed with shinyGO version 80 [37], shown are the top 20 GO terms). (F, G, H, I) qPCR analysis of selected genes of interest (based on A–C) in EV-stimulated and control male- (dots) and female-derived (triangles) monocytes. Rps9 was used as calibrator. ΔCq values were normalized to the median Cq value of pre-stimulation controls. (One-way ANOVA with Dunnett‘s or Šídák‘s multiple comparisons test, *p < 0.05, n = 5-6).
Fig 6.
Release of cytokines and myeloperoxidase upon EV stimulation.
Bone marrow-derived monocytes from male and female mice were stimulated for 24 h in vitro with 1000 EVs/cell or equal volume mock control. Supernatants of stimulated cells were analyzed by ELISA or flow cytometry-based multiplex cytokine assay (LEGENDplex). To control for the effect of protein denaturation on stimulatory capacity, EV and control samples were heat inactivated (h.i.) at 95°C for 10 min prior to stimulation. (A) Heatmap depicting median fluorescence intensity values for cytokines in supernatants of monocytes stimulated with EVs or controls as determined by LEGENDplex (IL-12p40, CXCL1, IL-6, TNF, IL-1β) or cytokine concentration as determined by ELISA (CCL3) (n = 3-6, depicted is the median of all samples of one condition). (B, C) Myeloperoxidase (MPO) concentration in supernatants of stimulated monocytes (B), bone marrow-derived neutrophils (C) and peripheral neutrophils (D) (isolated from spleen and blood, stimulated in the same manner as monocytes) as determined by ELISA. (One-way ANOVA with Šídák‘s multiple comparisons test, ** p < 0.01, **** p < 0.0001, n = 3-7).