Vesicle-based secretion in schistosomes: Analysis of protein and microRNA (miRNA) content of exosome-like vesicles derived from Schistosoma mansoni

Abstract

Exosomes are small vesicles of endocytic origin, which are released into the extracellular environment and mediate a variety of physiological and pathological conditions. Here we show that Schistosoma mansoni releases exosome-like vesicles in vitro. Vesicles were purified from culture medium by sucrose gradient fractionation and fractions containing vesicles verified by western blot analyses and electron microscopy. Proteomic analyses of exosomal contents unveiled 130 schistosome proteins. Among these proteins are common exosomal markers such as heat shock proteins, energy-generating enzymes, cytoskeletal proteins, and others. In addition, the schistosome extracellular vesicles contain proteins of potential importance for host-parasite interaction, notably peptidases, signaling proteins, cell adhesion proteins (e.g., integrins) and previously described vaccine candidates, including glutathione-S-transferase (GST), tetraspanin (TSP-2) and calpain. S. mansoni exosomes also contain 143 microRNAs (miRNA), of which 25 are present at high levels, including miRNAs detected in sera of infected hosts. Quantitative PCR analysis confirmed the presence of schistosome-derived miRNAs in exosomes purified from infected mouse sera. The results provide evidence of vesicle-mediated secretion in these parasites and suggest that schistosome-derived exosomes could play important roles in host-parasite interactions and could be a useful tool in the development of vaccines and therapeutics.

Figure 1

Overview of the procedure used for isolation and characterization of secreted exosome-like vesicles from Schistosoma mansoni. (A) Adult male and female worms were cultured 48-72 h in media containing exosome-depleted serum. Vesicles were purified from the culture media by differential centrifugation, followed by filtration through a 0.2 µm membrane and ultracentrifugation on a discontinuous 25, 30, 35% sucrose gradient, as described. The purification was monitored by western blot (WB) analysis, using antibodies against known exosomal markers (e.g. enolase) and electron microscopy (EM), prior to analyses of protein and miRNA content. (B) Schematic of the two major types of secretory extracellular vesicles. Membrane particles (or ectovesicles) are formed by outward budding of the plasma membrane. Exosomes are derived from the endocytic pathway via the formation of large multivesicular body (MVB) intermediates, which fuse with the plasma membrane releasing the vesicular cargo, exosomes, as well as other contents into an extracellular environment. Alternatively, the MVBs can be directed to lysosomes and degraded [see 24 for further details].

Figure 2

Purification of exosome-like vesicles from S. mansoni. Vesicles were collected from worm culture media and partially purified through differential centrifugation as shown in Fig. 1. The resulting crude vesicular pellet was resuspended in PBS, filter sterilized (0.2 µm filter) and subsequently fractionated on a discontinuous 10–50% sucrose gradient. Gradient fractions were tested for total protein content by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (A) and then western blotting (WB) with an antibody against enolase, a common exosomal marker (B). The results show enolase immunoreactivity between the 25% and 35% sucrose fractions. All subsequent purifications were performed by applying the filter-sterilized, crude vesicular pellets directly onto a discontinuous 25, 30, 35% sucrose gradient followed by ultracentrifugation, as described (C) Transmission electron microscopy analysis of purified exosome-like vesicles from S. mansoni. The scale bar indicates 100 nm.

Figure 3

Gene Ontology (GO) analysis of proteins recovered from S. mansoni exosome-like vesicles. The identified proteins analyzed with Blast2GO and were classified according to Biological Process (A), Cellular Component (B) and Molecular Function (D), as defined by the GO consortium.

Figure 4

Comparative analysis of S. mansoni microRNAs (miRNA) obtained from whole worms and purified exosome-like vesicles. The data are shown as the Log₂ ratio of normalized reads in the exosomal sample relative to the whole worm sample. Only the most abundant miRNAs are shown. Those miRNAs that are present at about the same level in the two samples, or are enriched in exosomes (Log₂ ≥ 0) are marked.

Figure 5

Quantitative qRT-PCR analysis of S. mansoni exosomal miRNAs in sera of infected mice. Circulating exosomes were purified from sera of S. mansoni –infected mice at 6–7 weeks post-infection or uninfected controls of the same age, using ExoQuick. RNA was extracted from the purified exosomes and then used for amplification of four S. mansoni miRNAs (Sma-mir-125a, Sma-mir-125b, Sma-mir-71a, Sma-bantam) by qRT-PCR. Data were median normalized relative to a “spike-in” synthetic miRNA^, and are shown as the fold-change relative to the uninfected control sample (background).