30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Prof.

A synergistic approach identifies key host factors required for apicomplexan parasites

Abstract text Apicomplexan parasites are obligate intracellular organisms responsible for major human and bovine infectious diseases, including malaria, toxoplasmosis and theileriosis. In this project we used a synergistic approach to identify host cell pathways that are essential for the parasite but dispensable for the host cell. By performing a genome-wide CRISPR/Cas9 drop out screen in Theileria-infected and non-infected bovine macrophages we obtained a list of 111 genes specifically essential for infected cells, with one third of these involved in metabolic pathways. We then combined genetic screens with chemoinformatic modelling, and ran gene knock-out simulations of these candidates in a human hepatocyte-Plasmodium falciparum metabolic model. Hydroxymethylbilane synthase (HMBS), an enzyme involved in heme biosynthesis, was predicted to be dispensable for uninfected hepatocytes but necessary for the survival of Plasmodium in the host cell. In order to confirm this prediction, we generated HMBS knockout clones in the human haploid cell line HAP1. Upon infection with the widely used rodent model organism Plasmodium berghei, we observed a significant decrease in parasite numbers, accompanied by a striking reduction in parasite liberation from infected cells. To exclude Cas9 off-target effects, we complemented HMBS and obtained a complete reversion of the phenotype. In conclusion, our results indicate that both Plasmodium and Theileria parasites depend on host HMBS for their survival. This suggests that Plasmodium, which expresses all enzymes of the heme pathway, not only relies on endogenous heme biosynthesis but also requires an intact host pathway for liver stage development.

Drug Development/Target Identification

Parasite-Host Interaction

Talk

IFN lambda protects mice from lethal oral T. gondii infection

Abstract text Toxoplasma gondii (T. gondii) is food-borne parasite that is an important cause of clinical disability in humans. Bradyzoites or sporozoites released upon rupture of tissue cysts or oocysts disseminate throughout the host. Interferons are essential for innate and adaptive immune responses against a wide variety of pathogens. Interferon lambda (IFN-λ) protects mucosal barriers during pathogen exposure. The intestinal epithelium is the first contact site for T. gondii with its hosts and the first defense line that limits parasite infection. Knowledge of very early T. gondii infection events in the gut tissue is limited and a possible contribution of IFN-λ has not been - to the best of our knowledge - investigated so far. Here, we demonstrate with systemic interferon lambda receptor (IFNLR1) and conditional (Villin-Cre) knockout (ko) mouse models of oral T. gondii infection and mouse intestinal organoids a significant impact of IFN-λ signalling in intestinal epithelial cells. Furthermore, a probe-based qPCR reveals increased parasite load in the ileum in ko compared with wt animals. Infective parasite stages cross the intestinal epithelial barrier to infect the lamina propria. In the lamina propria, T. gondii replicates in a variety of different cell types before disseminating throughout the host. Microsections of intestinal tissue demonstrate augmented T. gondii replication in the lamina propria of ko compared with wt animals. Besides intestinal epithelial cells, our results obtained with bone marrow-chimeras reveal an additional impact of IFN-λ signalling in neutrophils to parasite control. This study expands the repertoire of interferons that contribute to the control of T. gondii in mice and may lead to novel therapeutic approaches against this world-wide zoonotic pathogen.

Parasite Immunology

Prof., Dr.

Experimental infection of sheep at mid-pregnancy with archetypal type II and type III Toxoplasma gondii isolates exhibited different phenotypic traits

Abstract text Introduction The genotypic diversity of Toxoplasma gondii has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored Objectives To investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies Material &amp; methods Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n=33) and (ii) the lesions and parasite tropism and burden at 14- and 28-days post infection (dpi) (n=40) Results The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, fetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and fetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in fetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii-DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution Conclusions Genotype and phenotypic traits of each T. gondii isolate has an important role on the outcome of infection in ovine toxoplasmosis and mechanisms responsible for it need further investigations

Veterinary Parasitology

Toxoplasma gondii infection-driven DNA damage and chromosome instability in human host cells

Abstract text Introduction: The obligate intracellular apicomplexan parasite Toxoplasma gondii is a globally spread zoonotic protozoa causing severe health problems in humans and animals. Prenatal infections may cause abortion or severely affect progeny welfare, and acute T. gondii infections may become life-threatening in immunocompromised patients. Recently, we reported that T. gondii infection induces an increased percentage of binucleated cells, host cell chromosome segregation defects and cytokinesis failure in bovine endothelial cells, which also showed cell cycle arrest in S-phase, suggesting that parasite infection may directly interfere with host DNA synthesis or impair DNA quality. Objectives: Our aim here was to study if T. gondii infection induces host cell DNA damage. Materials &amp; methods: DNA was quantified via FACS-based approaches. DNA damage was studied via comet assays and immunofluorescence-based detection of typical markers for DNA damage foci (γH2AX), S- (PCNA) and G2-phase (Geminin). Protein expression of ataxia-telangiectasia mutated (ATM)- and ATM-and Rad3 (ATR)-related key markers was assessed by Western blotting. Intracellular and extracellular ROS was quantified via DCHF-DA- and AmplexRed assay. Results: T. gondii infection led to host cell S-phase arrest, binucleated cell phenotype and micronuclei formation already 15 min after the invasion. Both, comet assays and γH2AX detection revealed that T. gondii triggered DNA double-strand breaks shortly after infection and at a higher percentage in S-phase. However, ROS levels were not changed between 15-180 min p. i. The results showed that the DNA damage response (DDR)-, ATM-dependent pathway was activated in T. gondii-infected cells. Interestingly, effector molecules of the overall DDR pathway like phospho-p21 and cyclin E1 were also found enhanced after infection. To address the role of cyclin E in this process, a T. gondii mutant (RHΔhce1, RHΔmyr1), which fails to induce host cell cyclin E1 expression, was here used for infection. Overall, infections with mutated tachyzoites induced host cell DNA damage equal to control parasites, thus denying cyclin E-dependent reactions. Conclusion: Current data suggest that T. gondii triggers genome instability and DNA strand breaks early after infection. Both processes are neither dependent on host cellular ROS or cyclin E1 upregulation.

Prof

Characterisation of a disordered nuclear effector protein that interacts with FOXO1 in Theileria-transformed cells

Abstract text Theileria are tick-borne apicomplexan parasites that infect bovine leukocytes and cause Tropical Theileriosis. Within a few days of infection, parasitized leukocytes acquire a transformed phenotype comprising uncontrolled proliferation, resistance to apoptosis, immortality and increased invasiveness. This phenotype depends strictly on the presence of a viable parasite. RNA sequencing allowed us to identify extensive gene expression changes in primary macrophages following infection with Theileria. These include an upregulation of genes involved in DNA replication, cell cycle and translation, and a downregulation of genes involved in adhesion and innate immune response. To try to understand how Theileria modulates host gene expression networks, we used a comparative bioinformatics approach to identify Theileria-encoded effector proteins that are exported into the host nucleus. To facilitate this we performed Oxford Nanopore and Illumina whole genome sequencing on seven T. annulata clones isolated from different geographical regions. We analysed features such as selective pressure (dN/dS ratio) and protein disorderedness to identify and validate novel Theileria effector proteins. One of the identified proteins, TaC12_008960, is highly enriched in the host nucleus where it binds to the transcription factor Forkhead box protein O1 (FOXO1). FOXO transcription factors regulate many cellular processes including nutrient metabolism, DNA damage response, autophagy, cell cycle progression and oxidative stress response. FOXO1 expression is increased following infection, and immunofluorescence and western blot analysis confirmed that FOXO1 is localized in the nucleus of infected cells. We are exploring the functional significance of this interaction and testing what effect TaC12_008960 nuclear expression has on FOXO1 activity.

Molecular Parasitology

Theileria annulata NIDP2 belongs to a novel secreted protein family and interacts with the tumour suppressor STAG2 and the p53-regulating protein NOC2L

Abstract text Theileria annulata is a tick-transmitted apicomplexan parasite that causes a fatal cancer-like disease in the bovine host. The transient malignant phenotype is not based on genome instability or mutations at the DNA level as in cancer cells. We hypothesized that exported parasite proteins are responsible for the observed alteration of host signaling pathways in infected leukocytes. Only few exported proteins have been identified so far. To reveal uncharacterized Theileria effector proteins we used the TurboID proximity labeling technology and targeted the host cell nucleus and parasite membrane. By this approach we identified a novel repetitive protein locus of four intrinsically disordered proteins uniquely present in transformative T. annulata and absent in the non-transformative species T. orientalis. Co-immunoprecipitation experiments revealed an interaction of the exported protein NIDP2 with the host proteins NOC2L and STAG2. NOC2L is a protein involved in tumor suppressor p53 silencing. The STAG2 gene, another known tumor suppressor, encodes a subunit of the cohesin complex, which is involved in sister chromatid cohesion, with important roles in chromatin structure maintenance, gene expression and DNA repair. Further investigations are now being undertaken to analyze the contribution of these interactions to host cell transformation. The latest results will be presented at the conference. .

Session XVI
Parasite-Host Interactions 4 – Protozoa 2

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Vorsitzende

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Session XVIParasite-Host Interactions 4 – Protozoa 2

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Vorsitzende

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Session XVI
Parasite-Host Interactions 4 – Protozoa 2