Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

Abstract

Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence.

Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925).

Results: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs.

Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

Keywords: Malaria; Plasmodium falciparum; exosomes; extracellular vesicles; proteomics.

Figure 1.. Isolation of PfEVs.

A). Schematic showing the steps followed to purify PfEVs. B). Transmission electron microscopy was used to confirm the presence of vesicles in the pellet. The sizes ranged between 27–411nm (median= 132 nm and mean±SD = 143nm≥66). 73% were below 150nm and 90% were below 200 nm. C). Protein extract from each of the density gradient fraction was run on SDS-PAGE gel and stained with silver. The common band is albumin and lanes 9, 10 and 11 corresponding to fractions 10,11 and 12 seem to contain PfEV proteins. This gel only shows analysis of PfEVs from TR time point. The original uncropped image for both RT and TR time points is available at https://osf.io/wdg96/ .

Figure 2.. Virulence-associated parasite proteins are significantly enriched in PfEV proteome.

A). Gene Ontology enrichment analysis for cellular components (N=153). Plotted is the –log p-value (Bonferroni adjusted for 56 comparisons) against the GO terms. The horizontal red line indicates the cut-off for significance (p<0.01). The most significant GO terms were associated with ribosomal, exported and invasion proteins (apical complex). Note the rhoptry proteins (green bar, highlighted with blue line), especially those of the rhoptry bulb were significantly enriched. The rhoptry has been hypothesized to be the equivalent of the endosomal multivesicular body containing vesicles to be secreted to the extracellular environment as exosomes. Enrichment for genes related to intracellular organelles (purple bars) such as mitochondria, ER and nuclear was not significant. B). GO-terms associated with the top 50 most abundant proteins (#unique peptide≥4). Plotted is the –log p-value (Bonferroni adjusted for 38 comparisons) against the GO terms. Genes exported to the vesicular compartments within the cytosol of the parasitized red blood cells and those secreted from the endomembrane compartments of the merozoites such as the rhoptry and dense granules were significantly enriched.

Figure 3.. Exported and invasion related P. falciparum proteins form the core proteome of PfEVs.

A) Venn diagram showing that the proteome of PfEVs from the Kenya isolate had extensive overlap with a previously published PfEV proteome (Mantel et al. ). B) PfEV core proteins: GO-terms enrichment analysis for cellular components of the PfEV proteins common in both the Kenyan and the long-term laboratory isolates used in Mantel et al. . Plotted is the –log p-value (Bonferroni adjusted for 53 comparisons) against the GO terms. C) 9605 specific PfEVs proteins: GO-term enrichment analysis showing that the PfEV proteins specific to the Kenyan isolate. Plotted is the –log p-value (Bonferroni adjusted for 46 comparisons) against the GO terms. D) Venn diagram showing the overlap between the PfEV proteome identified in 1) this study (dark orange), 2) the previous PfEV proteome (Mantel et al. Cell Host Microbe 2013) (blue), 3) Schizont post rupture vesicles (Millholland et al. MCP 2011) (red), 4) plasma microparticles from patients with acute P. falciparum infection (Antwi-Baffour et al. proteome Sci 2017) (yellow).