Left panel: Interaction between PfVps60 and PfBro1. (a) STORM immunostaining detection of PfVps60 and PfBro1 in the blood stages of P. falciparum. (b) Field showing non-infected and infected RBCs. The inset contains a bright field low-resolution image to show the non-infected RBCs. Arrows pinpoint extracellular vesicles bound to non-infected RBCs, whose contours are indicated by dashed lines. Scale bars: 2 μm. Manders’ overlap coefficient demonstrated that PfBro1 and PfVps60 colocalized in the trophozoite stage of the intraerythrocytic cycle (A). Interestingly, vesicles labeled with PfBro1 and PfVps60 were detected bound to the surface of non-infected RBCs (B). Right panel: Effect on PfVps32 and PfBro1 by PfVps60 disruption. (a) Representative images of human erythrocytes infected with a P. falciparum KO-PfVps60 strain and fixed with 4% PFA to detect PfVps32 and PfBro1 proteins (green) and WGA (red) by indirect immunofluorescence. Cell nuclei were visualized with Hoechst 33342 (blue). Arrows show protein-labeled puncta adjacent to the membrane of the pRBCs. The arrowhead shows a vesicle-like structure labeled with PfBro1. For each protein are shown two microscope fields corresponding to the KO strain. Scale bars: 10 μm. To assess whether the observed EV reduction was only due to the effect of PfVps60 absence or to a generalized disruption of the ESCRT machinery, we evaluated the distribution of PfVps32 and PfBro1 in the KO line. As shown in A, both proteins appeared clustered around the periphery of the parasite, and in some cases large vesicular-like structures stained with PfBro1 were observed (A, arrowhead). However, some PfVps32- and PfBro1-labeled puncta were observed adjacent to the RBC plasma membrane suggesting their export to the RBC cytoplasmic space.

Avalos-Padilla Y, Georgiev VN, Lantero E, Pujals S, Verhoef R, N Borgheti-Cardoso L, Albertazzi L, Dimova R, Fernàndez-Busquets X. The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection. PLoS Pathog. 2021 17(4):e1009455. PMID: 33798247