Winners vs. losers: Schistosoma mansoni intestinal and liver eggs exhibit striking differences in gene expression and immunogenicity.

The eggs of the blood fluke Schistosoma mansoni are the main cause of the clinical manifestations of chronic schistosomiasis. After laying, the egg "winners" attach to the endothelium of the mesenteric vein and, after a period of development, induce the growth of a small granuloma, which facilitates their passage to the intestinal lumen. Egg "losers" carried by the bloodstream to non-specific tissues also undergo full development and induce large granuloma formation, but their life ends there. Although these trapped eggs represent a dead end in the parasite life cycle, the vast majority of studies attempting to describe the biology of the S. mansoni eggs have studied these liver-trapped "losers" instead of migrating intestinal "winners". This raises the fundamental question of how these eggs differ. With robust comparative transcriptomic analysis performed on S. mansoni eggs isolated 7 weeks post infection, we show that gene expression is critically dependent on tissue localization, both in the early and late stages of development. While mitochondrial genes and venom allergen-like proteins are significantly upregulated in mature intestinal eggs, well-described egg immunomodulators IPSE/alpha-1 and omega-1, together with micro-exon genes, are predominantly expressed in liver eggs. In addition, several proteases and protease inhibitors previously implicated in egg-host interactions display clear tissue-specific gene expression patterns. These major differences in gene expression could be then reflected in the observed different ability of liver and intestinal soluble egg antigens to elicit host immune responses and in the shorter viability of miracidia hatched from liver eggs. Our comparative analysis provides a new perspective on the biology of parasite's eggs in the context of their development and tissue localization. These findings could contribute to a broader and more accurate understanding of parasite eggs interactions with the host, which have historically been often restricted to liver eggs and sometimes inaccurately generalized.

Proteases and their inhibitors involved in Schistosoma mansoni egg-host interaction revealed by comparative transcriptomics with Fasciola hepatica eggs.

Schistosoma mansoni eggs are the main causative agents of the pathological manifestations of schistosomiasis. The eggs are laid in the host bloodstream, then they migrate through the intestinal wall into the lumen. However, a significant proportion of the eggs become lodged in the liver, where they cause inflammation and fibrosis. In this study, we focus on a specific group of proteins expressed by the egg, namely proteases and their inhibitors. These molecules are often involved in schistosome-host interactions, but are still unexplored in the egg stage. Using RNA-seq and comparative transcriptomics of immature and mature S. mansoni eggs, we mapped the portfolio of proteases and their inhibitors, and determined their gene expression levels. In addition, we compared these data with gene expression of proteases and their inhibitors in Fasciola hepatica eggs. Fasciola hepatica eggs served as a useful comparative model, as they do not migrate through tissues and inflict pathology. We detected transcription of 135 and 117 proteases in S. mansoni and F. hepatica eggs, respectively, with 87 identified as orthologous between the two species. In contrast, we observed only four orthologous inhibitors out of 21 and 16 identified in S. mansoni and F. hepatica eggs, respectively. Among others, we measured high and developmentally regulated levels of expression of metalloproteases in S. mansoni eggs, specifically aminopeptidase N1, endothelin-converting enzyme 1, and several leishmanolysin-like peptidases. We identified highly transcribed protease inhibitors serpin and alpha-2-macroglobulin that are unique to S. mansoni eggs, and antistasin-like inhibitor in F. hepatica eggs. This study provides new insights into the portfolio of proteases and inhibitors expressed by S. mansoni with potential roles in egg tissue migration, stimulation of angiogenesis, and interaction with host blood and immunity.

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes.

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.