30th Annual Meeting of the German Society for Parasitology

The Achilles' heel of the fox tapeworm? – Investigation of the threonine metabolism of Echinococcus multilocularis

Abstract text Introduction Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode of the fox tapeworm Echinococcus multilocularis. Novel treatment options are urgently needed. In vitro experiments showed strong scavenging of threonine by E. multilocularis metacestodes. Metacestodes express a threonine dehydrogenase (EmTDH) that is potentially a future drug target against AE, as human TDH is non-functional. Objectives We aimed to investigate the importance of threonine uptake and metabolism for E. multilocularis and to study whether threonine-degrading pathways can be targeted for the development of new drugs against the parasite. Quinazoline carboxamids (QCs) are potent inhibitors of mouse TDH and we aimed to test them against E. multilocularis. Material and methods 13C4 L-threonine and metabolites were traced in in vitro cultured metacestodes to give insights into the metabolization of threonine in E. multilocularis. In addition, we studied the effect of L-threonine on the growth of in vitro cultured metacestodes and on the vesicle formation of E. multilocularis primary cells. QCs were tested against E. multilocularis metacestodes via damage marker release assay and in enzymatic assays against EmTDH activity in crude extracts of metacestodes as well as recombinantly expressed EmTDH. Results Flux analysis with 13C4 L-threonine showed that E. multilocularis metacestodes take up threonine and metabolize it to glycine, which is indicative for a functional TDH pathway. Significantly increased growth was shown for threonine-treated metacestodes. Increased numbers of vesicles were formed upon treatment of primary cell cultures with threonine, whereas the threonine analogue 3-hydroxy norvaline reduced the number of vesicles. One QC inhibited EmTDH in an enzymatic assay using crude extract of metacestodes and it was toxic against E. multilocularis metacestodes. However, recEmTDH was not inhibited by any of the here tested QCs. Conclusion Flux analysis, growth assays and vesicle formation assays provided evidence that threonine metabolism is important for E. multilocularis and inhibiting this metabolism could provide new treatment options. Further investigation of the in vivo situation are needed. Novel QC derivatives will be designed for successful inhibition of EmTDH to be tested in the here established enzymatic assays for medium throughput screening of potential other inhibitors targeting the threonine metabolism of E. multilocularis.

Molecular Parasitology

Parasite-Host Interaction

Talk

Prof. Dr.

Prof.

Chromosomal integration of a reporter gene by RNA-guided Cas-enzymes into a predicted genomic safe-harbor site of Schistosoma mansoni

Abstract text "Introduction" Schistosoma mansoni, a zoonotic parasite, is characterized by a complex lifecycle that includes an invertebrate snail host and a vertebrate final host. Until now, no established protocol exists for the stable transformation of schistosomes. For investigating the function of a gene of interest (GOI), knock-out models are common for various organisms, but not yet available for trematodes. Until now, RNA interference (RNAi) has been proven as the most suitable method for functional gene characterization. However, RNAi efficiency varies, and it can lead to ectopic effects. CRISPR/Cas-based editing is a powerful tool for gene characterization. To make this technique accessible for trematode research, we tried to establish a protocol for editing a bioinformatically predicted genomic safe harbor site (GSH) of S. mansoni. GSHs are sites in the genome that allow the integration of new genetic material without negatively effecting genome integrity or gene expression. "Objectives" The aim of this project was to establish a CRISPR/Cas-based approach to generate transgenic S. mansoni. "Material and methods" For editing the identified GSH, a 5'C6-PEG10-modified construct encoding an eGFP reporter-gene driven by a strong native promoter was used as donor DNA. Cas-mediated integration of the transgene was performed by electroporation of eggs. To this end, ribonucleoprotein complexes (RNPs) were used consisting of the enzymes Cas9 or Cas12a and respective guide RNAs targeting the GSH. Integration of the transgene was examined by PCR. Reporter-gene expression was analysed by RT-PCR at the transcript level, and by confocal laser scanning microscopy to reveal eGFP expression. "Results" Using both Cas9 and Cas12a, we demonstrated the accessibility of the predicted GSH. Comparison of both Cas-enzymes revealed a significantly higher editing efficiency for Cas12a. Furthermore, we confirmed reporter-gene integration into the selected GSH using both RNPs formed by Cas9 or Cas12a. Finally, eGFP signals were detected demonstrating reporter gene expression in electroporated eggs and miracidia. "Conclusion" Successful editing of a predicted schistosomal GSH was shown as well as the expression of a genome-integrated eGFP reporter-gene. These results are proof of concept for a new genome-editing approach in S. mansoni. Our results suggest that both enzymes, Cas9 and Cas12a, are useful for editing the S. mansoni, with a higher efficiency for Cas12a.

Drug Development/Target Identification

Application of an RNA-seq-based method for the global analysis of self-cleaving ribozyme activity in Schistosoma mansoni

Abstract text Ribozymes are RNAs that catalyze chemical reactions. Some ribozymes, so-called self-cleaving ribozymes, cut their own sugar phosphate backbone at a specific position to enable their biological functions. In the bloodfluke Schistosoma mansoni thousands of self-cleaving ribozymes are predicted. As their analysis by classic biochemical approaches is tedious, we know little about their activity in vivo. We developed and validated a transcriptome-wide method, called cyPhyRNA-seq, to screen for active self-cleaving ribozymes in total RNA extracts. With this strategy, we successfully analyzed self-cleavage activity of more than 100 self-cleaving ribozymes from paired and unpaired schistosomes in a proof-of-principle study. We uncovered several intriguing ribozyme representatives: We found active ribozyme examples as part of mobile genetic elements such as Perere-3, W-elements and SINE retrotransposons. Furthermore, we found ribozyme sequences with mutations expected to inactivate them but that nonetheless function in vivo. For a representative of such ribozymes, we show in vitro evidence for a possible trans-cleavage mechanism, whereas all known natural self-cleaving ribozymes cleave in cis. Additionally, we identified differences in ribozyme activity between paired and unpaired schistosomes, suggesting a pairing-dependent expression and cleavage for some of the self-cleaving RNAs in Schistosoma. Lastly, we investigated a subset of the representatives in vitro using co-transcriptional cleavage assays and reverse transcription PCR to validate their cyPhyRNA-seq activity profile. Thus, cyPhyRNA-seq is crucial for the monitoring of ribozyme activity in organisms that harbor self-cleaving RNAs and it facilitates the investigation of self-cleavage activity under different conditions, such as stress or developmental stage. Differences in ribozyme activity between investigated conditions could point to novel biological roles of these RNAs and might even suggest that self-cleaving ribozyme activity is regulated. Lastly, the application of cyPhyRNA-seq to a ribozyme-rich organism such as Schistosoma may enable the discovery of novel self-cleaving ribozymes. These research endeavors contribute to our current understanding of the versatility of RNA and its biological significance and enable a global, functional screening approach.

Sex and Gender in Parasitology

Dr.rer.nat

Helminthic glutamate dehydrogenase-dependent PGE2 production in monocyte and microglia potentiates Treg development with distinct transcriptional and epigenetic profiles

Abstract text Introduction: Immunoregulation of inflammatory, infection-triggered processes in the brain constitutes a central mechanism to control devastating disease manifestations such as epilepsy. In neurocysticercosis (NCC), an inflammatory and clinically pleomorphic disease of the human brain, and most common cause of epilepsy in endemic regions, the disease severity strongly depends on the viability of the larval cyst of the pork tapeworm T. solium. Whereas dead cysts are often associated with epileptic manifestations, viable cysts in the brain mostly remain clinically silent by yet unknown mechanisms, potentially involving regulatory T cells (Tregs) in controlling inflammation. Objective: In this work, we aim to uncover the underlying mechanisms for this dichotomy, especially the nature of cyst products and mechanisms controlling the development of Tregs during asymptomatic NCC and inflammation during symptomatic NCC. Materials and Methods: Peripheral and brain immune cells from mice and healthy volunteers were pulsed with parasite viable, decaying cyst materials and the recombinant expressed cyst enzyme glutamate dehydrogenase GDH. Immune modulation and underlying mechanistic aspects were identified via adoptive transfer of cyst-treated DCs, and qPCR/FACS surrogate markers expression associated with LC/MS/MS profiling of eicosanoids and precursors and PGE2/IL-10 receptors antagonists. The mechanisms underlying Treg development and epigenetic landscape and transcriptional signatures associated with GDH-PGE2/IL-10-induced Tregs as well as FACS-sorted Tregs from NCC infected and healthy individuals were addressed via sequencing (ATACSeq, RNASeq). Results: We demonstrated that the enzyme GDH from parasite viable cyst instructs tolerogenic CD206+ monocytes and Iba-1lo microglia to release IL-10 and the lipid mediator PGE2. These act in concert via their respective receptors, converting naive CD4+ T cells into brain homing CD25hiFoxP3+CTLA-4+CCR6+CCR7+ Tregs with distinct transcriptional signatures (e.g. JAK-STAT pathway) as identified in asymptomatic NCC patients. Moreover, while viable cyst strongly upregulated IL-10 and PGE2 transcription in microglia leading to Treg development, dead cyst material lacking GDH enzyme induced proinflammatory non-phagocytic microglia and TGF-β as potential drivers of epilepsy. Conclusion: Harnessing the GDH-PGE2-IL-10 axis and targeting TGF-ß signaling may offer an important therapeutic strategy in inflammatory epilepsy and NCC.

Parasite Immunology

Towards a cell atlas for the liver fluke Fasciola hepatica

Abstract text The worldwide prevalent liver fluke Fasciola hepatica causes fascioliasis, a neglected tropical disease and zoonosis. Comprehensive knowledge on the parasite&quot;s cell types and cell-specific gene expression repertoire is missing to date. This kind of insight would take research on drug target genes, developmentally, and metabolically important genes to a next level. Recent technological advances, like the introduction of microfluidics in single-cell RNA sequencing (scRNA-seq) have greatly enhanced and may be transferred to multicellular parasites. Here, we provide the first whole-organism cell atlas of F. hepatica as a basis for expression analysis of genes on a single-cell level. We established a digestion protocol to obtain high-quality single-cell preparations from adult F. hepatica worms. Cells were then enriched by flow cytometry and finally 19.000 cells processed by 10X Genomics scRNA-seq technology. Cell clusters and marker genes were identified by using the software package Seurat in R. In-situ hybridization (ISH) was used to confirm the sites of gene expression. Gene ontology (GO) enrichment analysis revealed specific biological processes, molecular functions, or cellular components associated with the identified cell clusters. We successfully identified more than 17 cell clusters that represent distinct cell types, including gastrodermal cells expressing cathepsins, somatic stem cells (neoblasts) expressing nanos2, and neuronal cells. Marker genes for each cluster were identified and their expression confirmed by ISH. Some clusters were enriched for genes of important biological processes, like signal transduction in neuronal cells and proliferation in the neoblast cluster. The resolution of this dataset allowed uncovering potential lineages spanning from the central neoblast cluster into other clusters like the vitellarium, spermatogonia and the female germline. Additionally, we were also able to identify a tissue-specific expression of several protein kinase genes in the tegument as well as reproductive organs, a gene family that is well studied in other helminths with respect to druggable targets. We present the first transcriptome for the liver fluke F. hepatica on a single-cell resolution. This dataset can serve as treasure trove for the discovery and study of tissue-type and cell-type specific genes in this parasite.

Food-borne Parasitose

The Hippo pathway regulates Echinococcus stem cell function

Abstract text Introduction: Alveolar echinococcosis (AE) is a lethal zoonosis prevalent in the Northern Hemisphere and is caused by the metacestode larva of the fox tapeworm Echinococcus multilocularis. Growth of the Echinococcus metacestode within host organs is stem cell driven and almost unrestricted, leading to massive tumorous lesions. Organ size in metazoans is typically regulated by the Hippo pathway, the contribution of which to Echinococcus development has so far not been addressed. Objectives: To characterize EchinococcusHippo signaling components and their contribution to parasite development. Materials &amp; methods: We used bioinformatics to identify Echinococcus Hippo pathway components and in vitro cultivation systems, combined with inhibitor assays were used to study their role in Echinococcus proliferation and growth. Results: We identified Echinococcus orthologs of the kinases Hippo and Warts, the regulatory proteins Salvador and Mats, the co-activator Yorkie and the transcription factor Scalloped which are all expressed by differentiated cells and by stem cells in Echinococcus larvae. By applying the known Hippo inhibitors TRULI and XMU-MP-1 to metacestode vesicles and protoscoleces, we observed significant effects on larval morphology indicating alterations in stem cell proliferation. Particularly, XMU-MP-1 induced &quot;giant&quot; stem cells in metacestode vesicles, indicating direct influences of Hippo signaling on parasite mitosis. Furthermore, we show that RNAi knockdown of Echinoccocus yorkie induces vesicle formation by stem cells. Conclusions: Our data indicate that a complete Hippo-signaling pathway is present in E. multilocularis and contributes to proliferation dynamics of germinative stem cells, which are the decisive cell type in parasite growth within the host. Our data are relevant for understanding Echinococcus stem cell based developmental processes and may be exploited for the development of novel anti-parasitics.

Genetic structure and phylogeography of Echinococcus granulosus sensu stricto genotypes and potential role of buffalo-dog cycle in CE epidemiology in Pakistan

Abstract text Introduction Pakistan is a neglected endemic focus for Echinococcus granulosus sensu lato, a zoonotic parasite species complex with the ability to infect wide spectrum of hosts. Wide gaps exist in literature for etiological agents of cystic echinococcosis (CE) involved in epidemiological manifestation of this disease. Objectives The study explored main transmission routes of this disease through molecular characterization of hydatid cyst isolates. Materials &amp; methods The hydatid cysts were collected from the seven major cities, encompassing four provinces, of Pakistan. Molecular characterization of the isolates obtained from sheep (n = 35), goats (n = 26), cattle (n = 30) and buffalo (n = 30) was based on partial mitochondrial nad1 gene. Additionally, computation of different genetic diversity indices such as number of haplotypes (hn), nucleotide diversity (nd) and haplotype diversity (hd) was carried out. The four subpopulations of E. granulosus were also evaluated for neutrality indices and estimation of genetic differentiation and degree of gene flow by employing a pairwise fixation index (Fst). Results Two strains of E. granulosus sensu stricto, G1/G3, and their haplotypes (n = 9) were observed to be cycling in sympatry in the domestic ungulate populations. G3 genotype had higher prevalence (66.94%) in the hosts compared to G1 genotype (33.06%) which was not surprising, considering the large buffalo population in Pakistan. Elucidating local transmission patterns of E. granulosus sensu stricto, buffalo-dog cycle emerged as one of the dominant causes of G3 dispersal. Population diversity indices had overall moderate values of haplotype diversity (0.6285 ± 0.038) coupled with low nucleotide diversity (0.00271 ± 0.0002). Population demographics and genetic variability indices suggested expanding parasitic population in multiple host spectrum. Conclusion It was demonstrated that the E. granulosus s.s. population in Pakistan was highly differentiated from the other regions of the world. Furthermore, large population of buffaloes in this region and stronger affinity of G3 for this host reflect heterogeneity in host specificity and adaptability to local environmental conditions making buffalo-dog cycle as one of the major transmission routes in CE epidemic in Pakistan. Following other regional studies, where sheep-dog cycle is considered as the major source of disease, might not fit the epidemiological situation in South Asia.

Session IV
Molecular Parasitology II – Helminths

Termin

Vorsitzende

Programm

Session IVMolecular Parasitology II – Helminths

Termin

Vorsitzende

Programm

Session IV
Molecular Parasitology II – Helminths