30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

An infectious duo: The slender – stumpy success story

Abstract text The unicellular protozoan Trypanosoma brucei brucei, together with T. vivax and T. congolense, causes Animal African Trypanosomiasis (AAT) in livestock and wildlife in sub-Saharan Africa. These diseases are accompanied by a high degree of economic damage, affecting the low-income population severely. Since the parasite has a digenetic life cycle, the mammalian host is not the only organism it infects. Much of the T. brucei life cycle takes place in the arthropod vector, the tsetse fly (Glossina spp.). There are two forms of T. brucei in the circulation of an infected animal: the slender and stumpy bloodstream forms (bsf). The slender form proliferates, while the stumpy form is cell cycle-arrested. It is common knowledge that the stumpy forms rapidly differentiate to the first insect stage once being ingested by the tsetse fly. For more than a century it was assumed that the slender bsf dies in the tsetse midgut. It was only in 2021 that we showed that the slender bsf are perfectly capable of infecting the tsetse fly and going through the entire life cycle. Since quantitative experiments with vector infections are extremely complex, the question remained whether stumpy and slender forms are actually equally effective in colonising the tsetse fly. Therefore, we performed co-infection experiments with two differently fluorescent cell lines of T. brucei, one of which as slender form, the other as stumpy. These experiments were accompanied by in vitro analyses of the developmental transition of slender and stumpy trypanosomes to the insect stage using RNA-Seq. Furthermore, we tested if age of the flies makes a difference. It is thought that tsetse flies are less likely to get infected the older they become. Hence, infections of either slender or stumpy bsf were done with flies after their second or third feed. Altogether, we show that slender and stumpy bsf are equally successful in infecting the vector and going through the entire life cycle. In this process, slender forms do not become stumpy, but turn on specific genes to directly become the first insect stage (procyclic) in the midgut of the fly. Preliminary results also indicate that slender bsf, along with stumpy bsf, can also infect non-teneral flies. It can now be said without a doubt that the slender bsf of T. brucei are also able to infect the tsetse fly vector.

Molecular Parasitology

Parasite-Host Interaction

Talk

The 3D architecture of the Trypanosoma brucei endosomes reveals the structural basis for rapid plasma membrane recycling

Abstract text Trypanosoma brucei is an extracellular human and livestock pathogen that follows an effective strategy to evade the host immune system. This strategy relies on stochastical changes in the expression of variant surface glycoproteins (VSGs) that cover the entire cell surface of the parasite. Remarkably, trypanosomes recycle one cell surface equivalent within just 12 min and internalize surface bound antibodies within 2 min. Consequently, this parasite requires to have a highly effective endosomal sorting system. Currently, the endosomal system in T. brucei is described as distinct compartments that represent early, late and recycling endosomes. However, this model cannot explain the rapid rate of uptake and recycling in these organisms. Therefore, we analyzed the 3D architecture of the endosomal system. For this purpose, we used different electron tomography and immuno-electron microscopic methods, as well as immunofluorescence and structured illumination microscopy (SIM) to analyse the 3D anatomy of the endocytic apparatus in T. brucei. Electron tomograms show that the trypanosome endosomal system is more elaborate than previously thought and differs significantly from the mammalian endosome. Instead of being divided in distinct compartments it seems to consist of few continuous membrane systems. Antibodies against endosomal marker proteins (Rab5 – early endosomes, Rab7 – late endosome, Rab11 – recycling endosome) were used to perform in depth colocalization studies in wide-field microscopy. These analyzes indicate a high level of correlation between the different Rab proteins. To overcome the resolution limit of wide-field microscopy, super resolution microscopy and immuno-electron microscopy was used. All methods provide evidence for the existence of distinct functional endosomal subcompartments on the same membrane system, which questions the textbook picture of separate early, late and recycling endosomes. Our results suggest that plasma membrane recycling in trypanosomes happens through a quick journey within an &quot;endosomal highway&quot;, implying that its speed is diffusion-limited and not by trafficking through physically separated endosomal structures. Thus, we propose that the stream-lined architecture of the trypanosome endosomes is a consequence of the extreme and essential kinetics of plasma membrane recycling in these parasites.

PD Dr.

A quantitative mass spectrometry approach to identify novel proteins involved in macrophage infections by Leishmania parasites

Abstract text Leishmania are single-cell parasites with an intracellular life cycle stage in phagocytic cells of their vertebrate hosts. Transcriptomic analyses of host cell defense mechanisms and how the parasites respond to these processes are already available. Due to the prevalence of post-transcriptional regulation in kinetoplastids however, transcriptomes do not reflect the protein composition very well. Here we present a systematic comparative in vitro study of the infection process using a quantitative proteomics approach to uncover novel virulence factors. Murine bone marrow-derived macrophages (BMDMs) were infected with 3 different species: L. mexicana, L. major, L. infantum. Whole cell protein extracts of parasites and BMDMs were prepared at 7 timepoints post-infection (0.5-72h) and were analysed using quantitative mass spectrometry. Candidate proteins were knocked out in L. mexicana and infection experiments were carried out to discover novel mechanisms involved in parasite adaptation during macrophage infections. Evaluation of infection experiments were carried out using fluorescence microscopy. An ImageJ macro was scripted to process images and automatically determine the number of infected macrophages. We quantified the expression of approximately 2000 murine proteins in all 3 infection experiments, while 1500, 1000 and 1400 proteins were quantified for infections with L. mexicana, L. major and L. infantum, respectively. The number of differentially-expressed proteins during the time course of infection varied for each species (349, 133, and 287 proteins for L. mexicana, L. major and L. infantum, respectively). Our experiments identified many proteins putatively involved in the infection process, which were not found in previously published transcriptome analyses. For example, gene ontology enrichment showed 41 L. mexicana protein IDs involved in oxidoreductase activity. Additionally, we found 24 differentially-expressed proteins without any functional or structural annotation in the TriTryp database. Automated evaluation of infection experiments with two knockout cell lines showed reduced infectivity. Our mass spectrometry approach identified novel virulence factors in Leishmania infections, that were not detected in transcriptomic screens. Automated evaluation of infection experiments successfully showed strong phenotypes for two knockout cell lines. Candidate proteins are being further investigated to decipher their role during the infections processes.

Role of the CFTR in pathogenesis of Giardia duodenalis infections

Abstract text Introduction As a widespread protozoal parasite, Giardia duodenalis is a common cause for abdominal pain, malabsorption and diarrhea. Intestinal epithelial barrier defects are frequently observed in patients affected by the multifactorial disease giardiasis, which is characterized by &quot;leaky&quot; barriers due to the disturbance of the tight junction complex. While the exact mechanisms remain unknown, recent findings suggest a novel chain of events amounting in epithelial barrier breakdown, which starts with a disturbed ion homeostasis during the early infection phase. Amongst these intestinal ion transporters, the anion channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) has been shown to be severly affected upon infection regarding its gene expression as well as its function. Objectives We aim to shed light on the putative role of the CFTR channel in G. duodenalis infections by investigating its impact on pathogenesis at different infection stages and processes such as the early altered ion homeostasis, disruption of the tight junctional complex and cellular differentiation (goblet cell hyperplasia) during the late infection phase. Materials &amp; Methods Using the CRISPR/Cas9-technology, healthy intestinal organoids were converted into functionally impaired CFTR-mutants through insertion of the most common mutation (F508del) found in cystic fibrosis (CF) patients, a disease caused by mutations in the CFTR gene. For comparison of CFTR-impaired vs CFTR-functional organoids, mutant and wildtype were cultured in a compartmentalized transwell system and monitored regarding epithelial integrity throughout the infection period via transepithelial electrical resistance (TEER) measurements. Results Data from prior experiments show a robust transcriptional and functional downregulation of the CFTR ion transporter. Preliminary infection experiments with healthy and CF-patient derived intestinal organoids hint towards an increased prevalence of Clca1-positive (goblet) cells during the late infection phase in CF-derived cultures as compared to wildtype organoids. Conclusion Due to the significant downregulation of the CFTR channel during G. duodenalis infections and its pathogenic role in a variety of different diseases, we hypothesized that non-functionality of this important ion channel may benefit the parasite during infection. Experiments using CFTR-impaired organoids are still ongoing and will be discussed during the conference.

Biological and immunological characterization of Entamoeba histolytica-derived extracellular vesicles

Abstract text The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis. While 90 % of intestinal infections remain asymptomatic, 10 % result in invasive disease. In some cases, parasites can invade the mucosa and migrate to the liver via the bloodstream, where amebic liver abscesses (ALAs) are formed. ALAs are deadly to humans if left untreated. Interestingly, ALA formation occurs more frequently in men compared with women. Onset of invasive disease is the result of parasite pathogenicity factors on the one hand and an overshooting immune response involving infiltrating monocytes and neutrophils on the other. To gain a better understanding of the interaction between parasite and host immune system, we investigate extracellular vesicles (EVs) as putative modulators of the immune response. EVs were isolated from the supernatant of E. histolytica cultures using an ultracentrifugation-based approach. Biological characterization of the EVs was performed by proteomics, miRNA sequencing, nanoparticle tracking analysis and transmission electron microscopy. Two clonal cell lines differing in their pathogenicity were used for EV isolation. Analysis of the EV cargo revealed considerable differences between the two cell lines that might be relevant in the context of pathogenicity. Analysis of putative immunostimulatory properties of these EVs was performed in vitro on primary murine monocytes and neutrophils from both male and female mice. RNASeq, flow cytometry and cytokine profiling were implemented to characterize the immune cell response. Stimulation of primary immune cells with EVs of both cell lines resulted in increased expression of activation markers on the cell surface and an increased secretion of pro-inflammatory cytokines, both in male- and female-derived cells. Regulation of genes involved in immune response was also observed using RNASeq. Taken together, these data suggest an involvement of EVs in the host immune response to E. histolytica infection.

Parasite Immunology

Prof.

Applying high-resolution mass spectrometry imaging to visualize parasite-host interaction

Abstract text Introduction Parasite-host interaction is on a mutual relationship: Parasites depend on nutrients from their host, and the host organism fights against exploiting its resources, leading to inflammation. Both effects affect the lipid composition and distribution. High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) is a powerful tool to localize and identify lipids in biological samples. Objectives We aimed to visualize the impact of parasitic infection on the lipid profile of host tissue. We studied two different parasite-host models: Besnoitia besnoiti, forming cysts in bovine skin, and Schistosoma mansoni eggs, inducing granuloma formation in livers of hamsters as infection models. Materials &amp; Methods AP-SMALDI MSI was performed on an AP-SMALDI5-AF ion source (TransMIT GmbH, Giessen, Germany) coupled to a high-resolution orbital trapping mass spectrometer (Q Exactive, Thermo Fisher Scientific, Bremen, Germany), enabling high lateral resolution (≥5 um pixel size) to depict fine structures in the tissue and high mass accuracy (resolution R=240000 at m/z 200) to analyze hundreds of biomolecules in parallel. Cryosections (20 µm thick) of tissue samples were prepared using a cryomicrotome (HM 525, Thermo Fisher Scientific) and coated with matrix using a pneumatic sprayer (SMALDIprep, TransMIT GmbH) before analysis. Data were uploaded to the Metaspace platform, and analytes were annotated using LipidMaps. Statistical analyses led to a list of up- and down-regulated compounds. MS images of these ions were created, enabling manual assignment to specific, parasite-induced structures (e.g., cysts, granuloma). Results We found lipids characteristic for infection in parasite-host systems. For S. mansoni, specific lipids were found to be up- or downregulated in granuloma regions around the eggs. We even visualized very small features, such as Schistosoma eggs, inside the granuloma. Additionally, substructures within the granulomas were identified based on lipids with characteristic distributions. For B. besnoitia, specific lipids for cyst walls and cyst content were identified, resulting in mass spectrometric images resembling H&amp;E stained sections. Conclusion Using MSI, we analyzed parasites inside their host. This opens the door for future studies of different parasite-host systems, comparison of parasitic life stages, and analyzing drug-treated infected samples.

Session XIX
Parasite-Host-Interactions 5 – Protozoa 3

Appointment

Chairs

Programme

Session XIXParasite-Host-Interactions 5 – Protozoa 3

Appointment

Chairs

Programme

Session XIX
Parasite-Host-Interactions 5 – Protozoa 3