Abstract
Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.
Highlights
Data sets we have detailed the pathways involved with Extracellular vesicles (EVs) biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via endosomal sorting complexes required for transport (ESCRT)-dependent multivesicular body (MVB) formation in the tegumental syncytium before being shed from the apical plasma membrane
We found that F. hepatica secretes at least two subpopulations of EVs that differ according to size, cargo molecules and potential site of release from the parasite
Profiling the Total Secretome of Adult F. hepatica—Taking advantage of our recently reported F. hepatica genome and transcriptome data sets for the major intramammalian lifecycle stages [11], we were able to perform a definitive characterization of the secretions of adult F. hepatica
Summary
Experimental Design and Statistical Rationale—Western blots and fluorogenic assays were performed with at least two biological replicates and three technical triplicates. Each match was manually validated and confirmed using InterPro (http://www.ebi.ac.uk/interpro/) which detects the presence of conserved protein domains Expression of these identified genes across the F. hepatica lifecycle was investigated using the differential expression analysis of all the gene models identified within the genome. Analysis of Peptidase Activity in EVs—The soluble contents of F. hepatica EVs recovered following the 15,000 ϫ g and 120,000 ϫ g centrifugation of fluke culture media were isolated by osmotic shock. Auto-activation of the native procathepsin L was carried out by incubating the soluble extract from the 15,000 ϫ g vesicle pellet (50 g total protein) at 37 °C in 100 mM citrate phosphate buffer, pH 4.5, containing 1 mM DTT. The 15K vesicle pellet was resuspended in PBS and aliquots placed on formvar-coated grids and examined in a FEI (Philips) CM100 transmission electron microscope, operating at 100 keV
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