30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Geno- and phenotyping of the liver fluke (Fasciola hepatica) for comparative host-parasite genotype interaction studies

Parasite-Host Interaction

Veterinary Parasitology

Abstract text The liver fluke Fasciola hepatica is among the most important endoparasites in ruminants worldwide causing clinical symptoms and production losses. Anthelmintic treatment is widely implemented, but reports on resistance are increasing and the development of a vaccine has not been successful so far. This project aims at investigating the cattle-F. hepatica interaction by simultaneous modelling of host- and parasite-derived genomic, proteomic and glycomic data to unravel novel approaches for prevention and control of F. hepatica. To this end, Holstein-Frisian dairy cows from preselected herds are sampled at the abattoir and various infections traits were recorded. All liver flukes present in the bile ducts were measured, will be genotyped and variations in protein and glycan composition of the surface tegument are currently investigated via SDS-PAGE and immunoblot-based assays. Out of the sampled animals (n=156), 53 cows showed patent F. hepatica infections with a mean fluke count of 5.5 flukes/liver. Investigation of the surface tegument revealed minor variations between individual flukes and major differences in glycosylation patterns compared to published data from Ireland, suggesting considerable variations between fluke populations. Finally, all data will be considered simultaneously in genotype-by-genotype interaction analyses, which will provide new insights into host tolerance/resistance induction to unravel potential candidates for future vaccine development.

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PD Dr.

Winners vs. Losers – Comparative transcriptomic analysis of schistosoma mansoni mature and immature eggs from gut and liver

Molecular Parasitology

Abstract text Introduction The eggs of Schistosoma mansoni are the main cause of the clinical manifestations of schistosomiasis. It is important to note, however, that only the egg "losers" trapped in the host tissues, are responsible for these pathologies. After laying, the egg "winners", on the other hand, manage to attach to the endothelium of the mesenteric vein, and after a period of development, induce the growth of a granuloma which facilitates their passage through the intestinal wall to gut lumen. "Losers" carried with a blood stream to non-specific tissues also undergo full development and induce a granuloma formation, but their life ends there. Although trapped eggs represent a dead end in the life cycle, vast majority of transcriptomic and proteomic studies attempting to describe the biology of the eggs have studied these liver-trapped egg "losers" instead of gut-attached egg "winners". Objectives To assess if and how the gene expression of the egg is affected by the surrounding tissues. Materials &amp; Methods We isolated eggs from the liver and intestinal tissues of experimentally infected mice, divided eggs into mature and immature and compared their transcriptomic profiles. Results Our results clearly show that gene expression in S. mansoni eggs is critically dependent on tissue localization. In addition to the crucial differences between eggs derived from the two tissues, the expression profiles of liver-derived eggs are very similar regardless of their developmental stage, whereas gut-derived eggs show remarkable changes during their maturation. The most differentially expressed genes of interest are the Venom allergen-like proteins (VALs), which, together with proteases and protease inhibitors, are substantially more active in intestinal eggs. In stark contrast, IPSE/alpha-1, omega-1, as well as the majority of micro-exon genes (MEGs), which are often discussed as the primary weapons of the egg-winners, are, in fact, restricted to liver losers. Conclusion We argue that such differential expression of many important groups of molecules directly reflects the environment in which the egg is located. While in the case of the gut-derived eggs, who attach themselves to the cell wall, the up-regulated molecules probably represent the tools for successful passage to the external environment, in the case of the liver-derived eggs, the specific expression is more likely a response to host defence systems and thus reflects the failure of these individuals.

Long-term unisexual infection with Schistosoma mansoni in mice has the potential to boost the immune response to oviposition of challenge infection

Parasite Immunology

Abstract text Background: The complexity of Schistosoma spp. life cycle and their highly effective immune evasion strategies, makes vaccine development challenging. Unisexual infection, excluding immunomodulatory parasite eggs, offers a distinct perspective on host immune responses to both worm sexes and thus may provide a better understanding of complex immunological processes and identification of new targets for vaccine research. We have recently shown that in mice, a long-term unisexual infection with schistosomes leads to an unpolarized Th1/Th2 response associated with an abnormally enlarged spleen and diffuse hepatic inflammation. Aim: In this study, we analyzed whether unisexual worms can mate after 3 months of single sex infection and thus the Th2 response induced by oviposition can reverse or heal the described systemic inflammation. Methods: Mice were infected with male or female cercariae and reinfected with the opposite sex after 12 weeks for the same period. Unisexually infected mice and bisexually infected mice served as controls. At 24 weeks after initial infection, we histologically examined worm mating as evidenced by presence of eggs, infection-related pathology associated with eggs and characterization of liver fibrosis. We analyzed the immune status of the livers and spleens by using flow cytometry, recorded blood cell composition, and performed gene expression analysis of the livers. Results: We found that unisexual worms find each other, mate, and start oviposition even after 3 months. However, we observed more severe hepatic fibrosis in the reinfected groups compared to the bisexually infected group. Egg deposition has been associated with a typical Th2 immune response in the liver after reinfection, along with increased CD4+ T cell immune cells recruitment. Our results show that eggs are able to restore the Th1/Th2 immune imbalance of a previous unisexual infection. However, the organ damage caused by the unisexual worms does not subside, but rather provides the basis of egg-induced inflammation. Conclusion: Schistosomes can survive unpaired for a long time and cause severe organ damage. Given that schistosomes can mate several months after unisexual infection and then worm- and egg-related organ damage can accumulate, infection status without egg proof must be considered. Another critical issue is the tendency to crosshybridization of human schistosomes with livestock specific species, which can further contribute to the spread of the parasite.

Utilisation of "Omics" to unmask the interactions of adult male and female Schistosoma mansoni with their host

Abstract text Introduction: Schistosoma spp. are gonochoristic trematodes that cause one of the most devastating worm parasitoses in the world. To date, there is no effective vaccine to protect against the infection, and drug treatment with praziquantel has limited success in endemic areas. In the search for new therapeutic approaches to combat this disease, adult female schistosomes have so far been neglected because they have little direct contact with the environment and thus with the host during their mating with male worms. Objectives: Using "Omic" technologies, we aim to study the interactions between adult male and female Schistosoma mansoni and their host to find targets for new therapeutic strategies. Materials &amp; methods: First, we studied the structure of the tegument of male and female unpaired and paired schistosomes after contact with human serum as the host interface. Comparative analyses were performed by electron microscopy and immunohistochemistry. In addition, the tegument proteome of male and female unpaired and paired schistosomes was examined using a novel and highly sensitive workflow by LC-MS/MS analysis. In addition, the effect of circulating antigens from male and female schistosomes on the host immune response was investigated. For this purpose, the transcriptome and immune cell populations in the spleens of unisexually infected mice were analyzed. Results: After incubation in human serum, adult male and female schistosomes exhibited marked surface enlargement, and females showed shedding of their outer surface. Using proteomic analyses, we identified 1519 tegument proteins for male and female unpaired and paired schistosomes. We identified more proteins in male than in female worms, regardless of whether they were derived from mating or from unisexual infection. For transcriptomic analyses, 22,207 transcripts were examined in the spleens of unisexually infected mice. Principal component analysis showed clear clustering of experimental groups. Our studies suggest that male and female S. mansoni elicit an egg-independent, non-polarized Th1/Th2 immune response in the host, with males having a significantly greater immunoregulatory influence on gene regulation in the spleen and thus on the host. Conclusion: Our initial prelimiary findings need to be investigated to discover and understand new metabolic pathways and immunomodulatory mechanisms of adult worms to contribute to the development of new therapeutic strategies against schistosomiasis.

Egg secreted proteins: greater diversity than we thought

Abstract text Introduction: The eggs produced by Schistosoma mansoni adults are responsible for the pathology in the host tissues. Immature eggs bind to the endothelial surface and until that point, they are immunologically inert. When mature, attached eggs actively induce host inflammatory processes to pass from the vasculature endothelial tissue into the gut environment. They evoke granuloma tissue formation - a highly organized multicellular cluster enriched with immune cells. Many eggs are washed out from mesenteric veins mostly to the liver, where granuloma formations cause significant pathologies. Objectives: So far, little is known about the capability of the excretory/secretory products of eggs to stimulate these processes. Only a few secreted molecules have been experimentally studied and had their function revealed. So far, most of the attention has been devoted to egg-specific glycoproteins IPSE-1/alpha-1 and omega-1, among others promoting Th2 polarization. Nevertheless, the entire molecular composition of egg secretome and the roles of particular molecules remain unresolved. Materials &amp; methods: We are currently analyzing a portfolio of molecules produced by S. mansoni eggs by combining transcriptomic analysis and biochemical approaches. We focused on developed and undeveloped eggs isolated from the gut and liver tissue. Moreover, eggs of different S. mansoni geographical isolates are being analyzed in detail by bioinformatic pipeline in our lab as we have evidence that significant differences in the inflammatory reactions of rodent models are present. Results &amp; conclusion: Our preliminary data pointed out that gene expressions differ significantly based on the developmental stage of the eggs, tissue localization and S. mansoni strain. Our findings especially highlight two groups of proteins, the micro-exon gene (MEG) family and venom allergen-like (VAL) proteins, which are the most abundant molecules in egg transcriptome and their presence is stage and tissue localization dependent. Multi-isoform schistosome-specific proteins from MEG family encode some of the major secreted proteins with unknown functions with ~80% of the protein-coding region comprising short symmetric exons from 6-36 bp. VAL family shares a unique SCP/TAPS protein domain, which varies in length between 120 and 170 amino acids. Detailed immunohistochemical and biochemical studies are ongoing.

Session IX
Parasite-Host-Interactions I – Trematoda

Appointment

Chairs

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Session IXParasite-Host-Interactions I – Trematoda

Appointment

Chairs

Programme

Session IX
Parasite-Host-Interactions I – Trematoda