Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells

Abstract

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

Figure 1. Microvesicles are present at the surface of the Echinostoma caproni tegument.

Parasite tegumental area as seen by scanning electron microscopy (SEM) at different magnifications: ×200000 (A), and ×350000 (B). t: tegument; mv: microvesicles. The dots in the scale bars correspond to 1/10 of the length indicated in the figure.

Figure 2. Echinostoma caproni secretes exosome-like vesicles.

Production of E. caproni vesicles seen by transmission electron microscopy (TEM) at different magnifications: ×100000 (A), ×200000 (C), ×80000 (B, D). t: tegument; mvb; multivesicular bodies.

Figure 3. Exosome-like vesicles obtained from Echinostoma caproni and Fasciola hepatica.

Excretory/secretory materials (ESP) from E. caproni (A) and F. hepatica (B) were ultracentrifuged and the insoluble material was analyzed by transmission electron microscopy. Membranous vesicles of 30–100 nm of diameter are observed. Magnification ×200000.

Figure 4. Trematode exosomes-like vesicles contain typical ESP proteins.

E. caproni vesicles probed with preimmune sera (A). Immunodetection of enolase (B), actin (C) in E. caproni exosomes, and leucine aminopeptidase (LAP) in F. hepatica exosomes (D) are shown. Magnification ×100000.

Figure 5. E.caproni tegument presents microvesicles containing gold labeled actin.

t: tegument. Magnification ×80000.

Figure 6. Uptake of E. caproni exosomes by intestinal IEC-18 cells.

Confocal images of FM4–64 stained E. caproni exosomes (red) after different times of incubation. Magnification ×600. The scale bars correspond to 50 mm.