30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

The Physics of Parasitism (PoP-WS)

Parasite-Host Interaction

Abstract text Parasitism, the intimate antagonistic liaison between two species, has always fascinated scientists from a variety of disciplines. Traditionally, parasitology was concerned mainly with organismic studies, while today&quot;s parasitology focuses on medically-relevant cellular and molecular mechanisms, at ever-increasing depth. The DFG priority programme &quot;Physics of Parasitism&quot; (PoP) defines a new frontier in this field, namely the physics of parasites interacting with their hosts. Parasitism has evolved many times and hence, there are numerous convergent solutions to the challenge of how to physically hijack a host. These long periods of co-evolution have equipped parasites with high degrees of optimality. Examples are parasitic tools such as suckers and shields, or refined locomotive devices that allow navigation and also attachment in various body fluids, in crowded and confined spaces, and in highly viscous environments - often at surprisingly high speeds. &quot;Physics of Parasitism&quot; opens new chapters in both parasitology and the physics of life. The results obtained during the interdisciplinary endeavour will expose novel ways of combating parasitic diseases based on mechanobiology, against which resistances are unlikely to evolve. PoP focuses on three major physical/mechanical aspects of parasitism: (i) the construction and mechanical properties of the parasite bodies ("Bauplan"), (ii) the physics of parasite locomotion ("Locomotion"), and (iii) the physics behind mechanisms of host attachment ("Attachment"). In my lecture I will introduce and illustrate the concept of the "Physics of Parasitism" with examples.

Talk

Atomic force microscopy-based characterization of Giardia duodenalis attachment (POP-WS)

Molecular Parasitology

Abstract text Introduction Trophozoites of the parasitic protozoan Giardia duodenalis (GD) have a strong attaching behaviour on artificial surfaces and on the epithelial surface of the human duodenum, a well-known prerequisite for GD infections. Despite the profound knowledge about the cellular structures involved in attachment and the pivotal roles of the ventral disc, the lamellipodium-like ventrolateral flange and the beating flagellas, the quantitative adhesive forces of single trophozoites have not been reported yet. Objectives Here, the maximum detachment forces (MDF) of single GD trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) were determined. A direct comparison with the detachment behaviours of other eukaryotic species (i.e. Candida albicans yeast cells and human oral keratinocytes) was done to lay the foundation for a theoretical explanation of the attachment mode of GD. Materials &amp; Methods Fluidic force microscopy (FluidFM)-based single-cell force spectroscopy was used to quantify the adhesion parameters of single GD WB C6 trophozoites to glass. Briefly, surface-bound trophozoites were loaded with a FluidFM micropipette, simultaneously sucked by negative pressure and gradually pulled and removed from the surface by retraction of the micropipette. Recorded force-distance curves were evaluated for the MDF, the cell-detachment length and curve characteristics. For comparison, experiments with C. albicans ATCC 10231 and K2 keratinocytes were conducted. Results MDFs for GD trophozoites were with 8.2 ± 4.3 nN comparable to the values determined for C. albicans (7.5 ± 4.6 nN), but clearly lower than those seen with oral keratinocytes (55.8 ± 43.9 nN). Interestingly, our GD retraction curves displayed a gradual force increase on the pulled cell until the MDF was reached close to the cell-detachment length. For the other investigated species, however, a clear distance between the MDF and the cell-detachment length was observed with intermediate detachment forces probably caused by the rupture of single or few binding partners. Importantly, this pattern was never detected for GD trophozoites. Conclusion Our data indicate a unique adhesion mechanism for GD trophozoites on artificial surfaces that likely are dependent on the ventral disc contributing a major part to the overall adhesion force. Research with GD ventral disc mutants and with porous or nanorough surfaces is ongoing to confirm this hypothesis.

Univ.-Prof. Dr. rer. nat.

Investigation of Host-Parasite Interaction in Giardia muris infection via a fluorescent Ca2+ reporter - (POP-WS)

Parasite Immunology

Abstract text Introduction Infections with the protozoan parasite Giardia duodenalis cause a global health burden with over 200 million symptomatic cases every year. Giardia muris, a natural parasite in mice, serves as a model for the asymptomatic course seen in many cases of G. duodenalis infection. Our previous work suggests that modest adaptive immune responses operate together with the intestinal accumulation of pro-inflammatory myeloid cells, allowing for the control of infection in absence of overt immunopathology. Whether attachment of Giardia trophozoites directly drives immune defense by cells forming the intestinal epithelial barrier is not clear. Objectives As macrophages develop a pro-inflammatory phenotype upon activation of the mechano-sensitive Ca2+ channel PIEZO-1, we address whether attachment of G. muris to myeloid cells results in mechano-sensing driven defense mechanisms at the site of infection. Materials &amp; Methods Using 2-Photon microscopy, Bone Marrow Derived Macrophages (BMDM) from LysM-GCaMP6f reporter mice allow the visualization of PIEZO-1 induced Ca2+ influx based on fluorescence intensity. The performance of the system was tested by exposure of BMDM to Ca2+ ionophores, PIEZO-1 agonists and by direct mechanical stimulation applied by nanoindentation via Atomic Force Microscopy (AFM). Finally, BMDM were exposed to live G. muris trophozoites. Results Stimulation of BMDM with ionomycin caused an immediate increase in intracellular Ca2+, which was blocked by the addition of Ca2+ chelators. The application of the PIEZO-1 specific agonist YODA caused a similar effect, which could be reverted by the application of YODA antagonist GsMTx4. Furthermore, mechanostimulation of BMDM by nanoindentation via Atomic Force Microscopy initiated an increase in Ca2+ signal in the stimulated cells. Importantly, this finding was mirrored by a clear increase in Ca2+ reporter signal intensity in BMDM exposed to live G. muris trophozoites. Conclusions The LysM-GCaMP6f system reports the activation of mechanosensitive Ca2+ channels in myeoloid cells. Whether the attachment process of Giardia trophozoites results in the PIEZO-1 dependent activation of myeoloid cells and thereby triggers a local defense program against G. muris infection remains to be investigated.

Mrs.

Did protein engineering fail? Try biophysics! First structural insights into MEG-family proteins

One Health/NTD/Zoonoses

Abstract text Schistosomiasis is a parasitic disease caused by trematodes affecting the lives of millions of people worldwide1. Our work focuses on Schistosoma mansoni, the most widespread species of schistosomes infecting humans. Adult parasites reside in the circulatory system and lay eggs that need transiting from the endothelium of the blood vessel via host tissues into the gut lumen to be excreted to perpetuate the life cycle2. However, many of them remain trapped in host tissues, where they induce inflammation and chronic debilitating pathology. Eggs produce many bioactive molecules2,3, whose specific role is still yet to be characterized. We decided to focus on a significantly upregulated group of proteins named MEG proteins (micro-exon gene). These proteins are unique not only because of their genetic structure but also because they are specific to Schistosomatidae3. Due to alternative splicing, almost all members of the more than 30 families of MEGs generate several isoforms4. They do not show relevant homologies to other proteins in the UniProt database5, and their structure is still unresolved, as well as their specific role in egg-host interaction. We selected members of the MEG-2 and MEG-3 families. Our approach using standard protein engineering tools to produce recombinant proteins failed to obtain enough material. Therefore, we decided to divide the sequences into short synthetic overlapping peptides. In particular, we subdivided 3 isoforms of the MEG-2.1 family into 8 peptides. We collected a series of 2D NMR homonuclear and heteronuclear spectra, measured in the natural abundance of 13C and 15N. The attribution of the peaks in the 2D spectra, in combination with distance geometry and energy minimization software, allowed us to determine the structures of these peptides belonging to 2 out of the 3 isoforms. Here we present the first structural information on S. mansoni MEG proteins, which can serve to infer the structure of other MEG family members. Resolving the structure is the first step toward the elucidation of MEGs role in host-parasite interactions. 1. WHO. Schistosomiasis. 2022 https://www.who.int/news-room/fact-sheets/detail/schistosomiasis 2. Costain AH et al. Front immunol 2018, 9: 3042. 3. Li XH et al. PLoS NTD 2018; 12.2: e0006235. 4. DeMarco R, et al. Genome Res. 2010; 20(8):1112-21. 5. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49.D1: D480-D489.

Visualizing the surface dynamics on and in Trypanosoma brucei (POP-WS)

Abstract text Due to its extracellular lifestyle, Trypanosoma brucei is always exposed to the immune system of its host. While being able to switch their predominant variant surface antigen (VSG), their immune evasion strategy also heavily relies on the mobility of their surface coat, which enables them to remove antibodies from their cell surface within seconds via endocytosis. As the fast turnover on the surface must be mirrored in speed for degradation or recycling, we now also turn our attention to the inside of the cell. We want to visualize the dynamics inside the endosomal network in three dimensions to elucidate how the sorting of membrane proteins is realized in a system, that does not have the time for expensive membrane budding and fusion process. To measure the intracellular dynamics, we express an insect-stage specific membrane protein (EP1) in the bloodstream form of the parasite, which will stay confined to the endosomal compartments and the flagellar pocket. To selectively mark the protein, we Halo-tagged it. Living cells are labelled using picomoles of the dye, immobilized in hydro gel and imaged at 37°C. With the help of an astigmatic lens, we collect 3D information of our single emitters. The acquired data is processed using SMAP by Jonas Ries and reveals a resolution of 30 nm in x/y and 80 nm in z imaging at 25 Hz. The calculated positions of single emitters are then used to generate tracks using Swift by Ulrike Endersfelder. Those tracks are then analyzed using our custom software based on the SPTAnalysis by Pierre Parutto. Preliminary data of Atto643-NHS labelled VSG shows that the flagellar membrane can act as a diffusional barrier that can not easily be crossed by surface molecules and that an accumulation of signal could be localized inside the cell in proximity to the flagellar pocket. Overall, the surface dynamics of the VSG of the 3D data seem to be comparable with previously acquired 2D data. As the comparison of 2D and 3D data lead to a similar diffusion coefficient, the overall workflow for 3D data seems to be suitable. A challenge will arise with the spatially quite confined endosomal system to generate sufficient data for analysis, which will be compensated for by the long-lived Janelia Fluor dyes. In addition, our workflow would allow statistical analysis of the 3D track data, which could change the general understanding of the endocytotic flow and shed light on how the sorting and recycling mechanism really work.

Prof.

Optimal alignment of plasmodium merozoites at erythrocytes (POP-WS)

Abstract text Invasion of plasmodium merozoites into erythrocytes is an essential step for their survival and multiplication. The parasites align their apex with the membrane surface for successful invasion. It has been hypothesized that they use erythrocyte membrane deformability to efficiently align themselves. We use computer simulations to test the hypothesis. We model the merozoite as a hard egg-shaped particle and the erythrocytes using a continuum membrane model [1, 2]. Typical snapshot from computer simulations is shown in Figure 1. In this talk, I will discuss the role of erythrocyte deformability and parasite-membrane adhesion in the alignment process using multiscale modeling. We show that parasite-membrane interactions with an inhomogeneous contact interaction that describes an adhesion-strength gradient on the parasite plasma membrane lead to much faster alignment compared with experimentally measured timescales; pure rotational diffusion of the parasite leads to too slow alignment [3]. Therefore, we simulate the adhesion without a gradient but considering receptor-ligand bond dynamics [4]. I will show how the shape fluctuations and deformability of the erythrocytes along with the stochastic nature of receptor-ligand bond formation predict alignment times that are consistent with experimental observations. I will also discuss the role of parasite shape in the alignment process and show that original shape of the parasite performs better compared other shapes at various conditions [5]. Finally, I will provide an update on and outlook for our project on Toxoplasma gondii invasion into host cells within the SPP 2332 &quot;Physics of Parasitism&quot;. References: [1] Fedosov, Dmitry, et al. "A multiscale red blood cell model with accurate mechanics, rheology, and dynamics." Biophysical journal 98.10 (2010): 2215-2225. [2] Dasgupta, Sabyasachi, et al. "Membrane-wrapping contributions to malaria parasite invasion of the human erythrocyte." Biophysical journal 107.1 (2014): 43-54. [3] Hillringhaus, Sebastian, et al. "Importance of erythrocyte deformability for the alignment of malaria parasite upon invasion." Biophysical journal 117.7 (2019): 1202-1214. [4] Hillringhaus, Sebastian, et al. "Stochastic bond dynamics facilitates alignment of malaria parasite at erythrocyte membrane upon invasion." Elife 9 (2020): e56500. [5] Dasanna, Anil K., et al. "Effect of malaria parasite shape on its alignment at erythrocyte membrane." Elife 10 (2021): e68818.

Biophysics of Plasmodium sporozoite migration //POP-WS

Abstract text Plasmodium sporozoites are the forms of the malaria parasites transmitted by mosquitoes. They are slender, slightly curved cells that can migrate at high speed. While a number of proteins are known to be essential for migration, we have only a very limited understanding of the interplay between these. In order to migrate sporozoites need to attach to a substrate, generate force onto the substrate and to move forward and they have to detach the adhesive sites from the substrate. We have used a number of imaging and biophysics assays along with the generation of mutant parasites and will present some of these, such a laser trap and traction force experiments to measure force as well as 3D electron tomography to investigate the cytoskeleton of the parasite. Currently we are investigating the role of chiral elements in the migration of the parasite using a mix of in silico modelling, expansion microscopy and analysis of mutant parasites. These mutants lack actin or microtubule binding proteins that lead to non-chiral motility of the sporozoites.

Workshop IV
PoP

Appointment

Chairs

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Workshop IVPoP

Appointment

Chairs

Programme

Workshop IV
PoP