Dokument-ID Dokumenttyp Verfasser/Autoren Herausgeber Haupttitel Abstract Auflage Verlagsort Verlag Erscheinungsjahr Seitenzahl Schriftenreihe Titel Schriftenreihe Bandzahl ISBN Quelle der Hochschulschrift Konferenzname Quelle:Titel Quelle:Jahrgang Quelle:Heftnummer Quelle:Erste Seite Quelle:Letzte Seite URN DOI Abteilungen OPUS4-35000 Wissenschaftlicher Artikel Engstler, Markus; Beneke, Tom Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries. 2023 eLife 12 urn:nbn:de:bvb:20-opus-350002 10.7554/eLife.85605 Theodor-Boveri-Institut für Biowissenschaften OPUS4-29756 Wissenschaftlicher Artikel Rackevei, Antonia S.; Borges, Alyssa; Engstler, Markus; Dandekar, Thomas; Wolf, Matthias About the analysis of 18S rDNA sequence data from trypanosomes in barcoding and phylogenetics: tracing a continuation error occurring in the literature The variable regions (V1-V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549-561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system). 2022 Biology 11 11 urn:nbn:de:bvb:20-opus-297562 10.3390/biology11111612 Theodor-Boveri-Institut für Biowissenschaften OPUS4-27302 Wissenschaftlicher Artikel Krüger, Timothy; Maus, Katharina; Kreß, Verena; Meyer-Natus, Elisabeth; Engstler, Markus Single-cell motile behaviour of Trypanosoma brucei in thin-layered fluid collectives We describe a system for the analysis of an important unicellular eukaryotic flagellate in a confining and crowded environment. The parasite Trypanosoma brucei is arguably one of the most versatile microswimmers known. It has unique properties as a single microswimmer and shows remarkable adaptations (not only in motility, but prominently so), to its environment during a complex developmental cycle involving two different hosts. Specific life cycle stages show fascinating collective behaviour, as millions of cells can be forced to move together in extreme confinement. Our goal is to examine such motile behaviour directly in the context of the relevant environments. Therefore, for the first time, we analyse the motility behaviour of trypanosomes directly in a widely used assay, which aims to evaluate the parasites behaviour in collectives, in response to as yet unknown parameters. In a step towards understanding whether, or what type of, swarming behaviour of trypanosomes exists, we customised the assay for quantitative tracking analysis of motile behaviour on the single-cell level. We show that the migration speed of cell groups does not directly depend on single-cell velocity and that the system remains to be simplified further, before hypotheses about collective motility can be advanced. 2021 The European Physical Journal E 44 3 urn:nbn:de:bvb:20-opus-273022 10.1140/epje/s10189-021-00052-7 Theodor-Boveri-Institut für Biowissenschaften OPUS4-27028 Wissenschaftlicher Artikel Hempelmann, Alexander; Hartleb, Laura; van Straaten, Monique; Hashemi, Hamidreza; Zeelen, Johan P.; Bongers, Kevin; Papavasiliou, F. Nina; Engstler, Markus; Stebbins, C. Erec; Jones, Nicola G. Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells. 2021 Cell Reports 37 5 urn:nbn:de:bvb:20-opus-270285 10.1016/j.celrep.2021.109923 Theodor-Boveri-Institut für Biowissenschaften OPUS4-26387 Wissenschaftlicher Artikel Bakari-Soale, Majeed; Ikenga, Nonso Josephat; Scheibe, Marion; Butter, Falk; Jones, Nicola G.; Kramer, Susanne; Engstler, Markus The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process. 2021 Scientific Reports 11 1 urn:nbn:de:bvb:20-opus-263872 10.1038/s41598-021-97020-0 Theodor-Boveri-Institut für Biowissenschaften OPUS4-26174 Wissenschaftlicher Artikel Schuster, Sarah; Lisack, Jaime; Subota, Ines; Zimmermann, Henriette; Reuter, Christian; Mueller, Tobias; Morriswood, Brooke; Engstler, Markus Unexpected plasiticty in the life cycle of Trypanosoma brucei African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host's circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia. 2021 eLife 10 urn:nbn:de:bvb:20-opus-261744 10.7554/eLife.66028.sa2 Theodor-Boveri-Institut für Biowissenschaften OPUS4-24925 Wissenschaftlicher Artikel Borges, Alyssa R.; Link, Fabian; Engstler, Markus; Jones, Nicola G. The Glycosylphosphatidylinositol Anchor: A Linchpin for Cell Surface Versatility of Trypanosomatids The use of glycosylphosphatidylinositol (GPI) to anchor proteins to the cell surface is widespread among eukaryotes. The GPI-anchor is covalently attached to the C-terminus of a protein and mediates the protein's attachment to the outer leaflet of the lipid bilayer. GPI-anchored proteins have a wide range of functions, including acting as receptors, transporters, and adhesion molecules. In unicellular eukaryotic parasites, abundantly expressed GPI-anchored proteins are major virulence factors, which support infection and survival within distinct host environments. While, for example, the variant surface glycoprotein (VSG) is the major component of the cell surface of the bloodstream form of African trypanosomes, procyclin is the most abundant protein of the procyclic form which is found in the invertebrate host, the tsetse fly vector. Trypanosoma cruzi, on the other hand, expresses a variety of GPI-anchored molecules on their cell surface, such as mucins, that interact with their hosts. The latter is also true for Leishmania, which use GPI anchors to display, amongst others, lipophosphoglycans on their surface. Clearly, GPI-anchoring is a common feature in trypanosomatids and the fact that it has been maintained throughout eukaryote evolution indicates its adaptive value. Here, we explore and discuss GPI anchors as universal evolutionary building blocks that support the great variety of surface molecules of trypanosomatids. 2021 Frontiers in Cell and Developmental Biology 9 urn:nbn:de:bvb:20-opus-249253 10.3389/fcell.2021.720536 Theodor-Boveri-Institut für Biowissenschaften OPUS4-24468 Wissenschaftlicher Artikel Link, Fabian; Borges, Alyssa R.; Jones, Nicola G.; Engstler, Markus To the Surface and Back: Exo- and Endocytic Pathways in Trypanosoma brucei Trypanosoma brucei is one of only a few unicellular pathogens that thrives extracellularly in the vertebrate host. Consequently, the cell surface plays a critical role in both immune recognition and immune evasion. The variant surface glycoprotein (VSG) coats the entire surface of the parasite and acts as a flexible shield to protect invariant proteins against immune recognition. Antigenic variation of the VSG coat is the major virulence mechanism of trypanosomes. In addition, incessant motility of the parasite contributes to its immune evasion, as the resulting fluid flow on the cell surface drags immunocomplexes toward the flagellar pocket, where they are internalized. The flagellar pocket is the sole site of endo- and exocytosis in this organism. After internalization, VSG is rapidly recycled back to the surface, whereas host antibodies are thought to be transported to the lysosome for degradation. For this essential step to work, effective machineries for both sorting and recycling of VSGs must have evolved in trypanosomes. Our understanding of the mechanisms behind VSG recycling and VSG secretion, is by far not complete. This review provides an overview of the trypanosome secretory and endosomal pathways. Longstanding questions are pinpointed that, with the advent of novel technologies, might be answered in the near future. 2021 Frontiers in Cell and Developmental Biology 9 urn:nbn:de:bvb:20-opus-244682 10.3389/fcell.2021.720521 Theodor-Boveri-Institut für Biowissenschaften OPUS4-23064 Wissenschaftlicher Artikel Kramer, Susanne; Meyer-Natus, Elisabeth; Stigloher, Christian; Thoma, Hanna; Schnaufer, Achim; Engstler, Markus Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans. 2021 Nucleic Acids Research 49 3 urn:nbn:de:bvb:20-opus-230647 10.1093/nar/gkaa1142 Theodor-Boveri-Institut für Biowissenschaften OPUS4-17136 Wissenschaftlicher Artikel Hartel, Andreas J.W.; Glogger, Marius; Jones, Nicola G.; Abuillan, Wasim; Batram, Christopher; Hermann, Anne; Fenz, Susanne F.; Tanaka, Motomu; Engstler, Markus N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed. 2016 Nature Communications 7 urn:nbn:de:bvb:20-opus-171368 10.1038/ncomms12870 Theodor-Boveri-Institut für Biowissenschaften OPUS4-17770 Wissenschaftlicher Artikel Goos, Carina; Dejung, Mario; Wehman, Ann M.; M-Natus, Elisabeth; Schmidt, Johannes; Sunter, Jack; Engstler, Markus; Butter, Falk; Kramer, Susanne Trypanosomes can initiate nuclear export co-transcriptionally The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes. 2019 266-282 Nucleic Acids Research 47 1 urn:nbn:de:bvb:20-opus-177709 10.1093/nar/gky1136 Theodor-Boveri-Institut für Biowissenschaften OPUS4-17594 Wissenschaftlicher Artikel Krüger, Timothy; Engstler, Markus The fantastic voyage of the trypanosome: a protean micromachine perfected during 500 million years of engineering The human body is constantly attacked by pathogens. Various lines of defence have evolved, among which the immune system is principal. In contrast to most pathogens, the African trypanosomes thrive freely in the blood circulation, where they escape immune destruction by antigenic variation and incessant motility. These unicellular parasites are flagellate microswimmers that also withstand the harsh mechanical forces prevailing in the bloodstream. They undergo complex developmental cycles in the bloodstream and organs of the mammalian host, as well as the disease-transmitting tsetse fly. Each life cycle stage has been shaped by evolution for manoeuvring in distinct microenvironments. Here, we introduce trypanosomes as blueprints for nature-inspired design of trypanobots, micromachines that, in the future, could explore the human body without affecting its physiology. We review cell biological and biophysical aspects of trypanosome motion. While this could provide a basis for the engineering of microbots, their actuation and control still appear more like fiction than science. Here, we discuss potentials and challenges of trypanosome-inspired microswimmer robots. 2018 63 Micromachines 9 2 urn:nbn:de:bvb:20-opus-175944 10.3390/mi9020063 Theodor-Boveri-Institut für Biowissenschaften OPUS4-14274 Wissenschaftlicher Artikel Schwede, Angela; Jones, Nicola; Engstler, Markus; Carrington, Mark The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes In the mammalian host, the Trypanosoma brucei cell surface is covered with a densely packed protein coat of a single protein, the variant surface glycoprotein (VSG). The VSG is believed to shield invariant surface proteins from host antibodies but there is limited information on how far antibodies can penetrate into the VSG monolayer. Here, the VSG surface coat was probed to determine whether it acts as a barrier to binding of antibodies to the membrane proximal VSG C-terminal domain. The binding of C-terminal domain antibodies to VSG221 or VSG118 was compared with antibodies recognising the cognate whole VSGs. The C-terminal VSG domain was inaccessible to antibodies on live cells but not on fixed cells. This provides further evidence that the VSG coat acts as a barrier and protects the cell from antibodies that would otherwise bind to some of the other externally disposed proteins. 2011 201-204 Molecular & Biochemical Parasitology 175 2 urn:nbn:de:bvb:20-opus-142746 10.1016/j.molbiopara.2010.11.004 Theodor-Boveri-Institut für Biowissenschaften OPUS4-14081 Wissenschaftlicher Artikel Uppaluri, Sravanti; Nagler, Jan; Stellamanns, Eric; Heddergott, Niko; Herminghaus, Stephan; Pfohl, Thomas; Engstler, Markus Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional Microorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages. 2011 e1002058 PLoS Computational Biology 7 6 urn:nbn:de:bvb:20-opus-140814 10.1371/journal.pcbi.1002058 Theodor-Boveri-Institut für Biowissenschaften OPUS4-14461 Wissenschaftlicher Artikel Alizadehrad, Davod; Krüger, Timothy; Engstler, Markus; Stark, Holger Simulating the complex cell design of Trypanosoma brucei and its motility The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and predict a flagellar course we have not been able to measure in experiments so far. 2015 e1003967 PLOS Computational Biology 11 1 urn:nbn:de:bvb:20-opus-144610 10.1371/journal.pcbi.1003967 Theodor-Boveri-Institut für Biowissenschaften OPUS4-15866 Wissenschaftlicher Artikel Schuster, Sarah; Krüger, Timothy; Subota, Ines; Thusek, Sina; Rotureau, Brice; Beilhack, Andreas; Engstler, Markus Developmental adaptations of trypanosome motility to the tsetse fly host environments unravel a multifaceted in vivo microswimmer system The highly motile and versatile protozoan pathogen Trypanosoma brucei undergoes a complex life cycle in the tsetse fly. Here we introduce the host insect as an expedient model environment for microswimmer research, as it allows examination of microbial motion within a diversified, secluded and yet microscopically tractable space. During their week-long journey through the different microenvironments of the fly´s interior organs, the incessantly swimming trypanosomes cross various barriers and confined surroundings, with concurrently occurring major changes of parasite cell architecture. Multicolour light sheet fluorescence microscopy provided information about tsetse tissue topology with unprecedented resolution and allowed the first 3D analysis of the infection process. High-speed fluorescence microscopy illuminated the versatile behaviour of trypanosome developmental stages, ranging from solitary motion and near-wall swimming to collective motility in synchronised swarms and in confinement. We correlate the microenvironments and trypanosome morphologies to high-speed motility data, which paves the way for cross-disciplinary microswimmer research in a naturally evolved environment. 2017 e27656 eLife 6 urn:nbn:de:bvb:20-opus-158662 10.7554/eLife.27656 Medizinische Klinik und Poliklinik II OPUS4-13517 Wissenschaftlicher Artikel Harrington, John M.; Scelsi, Chris; Hartel, Andreas; Jones, Nicola G.; Engstler, Markus; Capewell, Paul; MacLeod, Annette; Hajduk, Stephen Novel African Trypanocidal Agents: Membrane Rigidifying Peptides The bloodstream developmental forms of pathogenic African trypanosomes are uniquely susceptible to killing by small hydrophobic peptides. Trypanocidal activity is conferred by peptide hydrophobicity and charge distribution and results from increased rigidity of the plasma membrane. Structural analysis of lipid-associated peptide suggests a mechanism of phospholipid clamping in which an internal hydrophobic bulge anchors the peptide in the membrane and positively charged moieties at the termini coordinate phosphates of the polar lipid headgroups. This mechanism reveals a necessary phenotype in bloodstream form African trypanosomes, high membrane fluidity, and we suggest that targeting the plasma membrane lipid bilayer as a whole may be a novel strategy for the development of new pharmaceutical agents. Additionally, the peptides we have described may be valuable tools for probing the biosynthetic machinery responsible for the unique composition and characteristics of African trypanosome plasma membranes. 2012 e44384 PLoS One 7 9 urn:nbn:de:bvb:20-opus-135179 10.1371/journal.pone.0044384 Theodor-Boveri-Institut für Biowissenschaften OPUS4-15823 Wissenschaftlicher Artikel Zimmermann, Henriette; Subota, Ines; Batram, Christopher; Kramer, Susanne; Janzen, Christian J.; Jones, Nicola G.; Engstler, Markus A quorum sensing-independent path to stumpy development in Trypanosoma brucei For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival. 2017 e1006324 PLoS Pathogens 13 4 urn:nbn:de:bvb:20-opus-158230 10.1371/journal.ppat.1006324 Theodor-Boveri-Institut für Biowissenschaften OPUS4-13459 Wissenschaftlicher Artikel Heddergott, Niko; Krüger, Timothy; Babu, Sujin B.; Wei, Ai; Stellamanns, Erik; Uppaluri, Sravanti; Pfohl, Thomas; Stark, Holger; Engstler, Markus Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy. 2012 e1003023 PLoS Pathogens 8 11 urn:nbn:de:bvb:20-opus-134595 10.1371/journal.ppat.1003023 Theodor-Boveri-Institut für Biowissenschaften OPUS4-14651 Wissenschaftlicher Artikel Bargul, Joel L.; Jung, Jamin; McOdimba, Francis A.; Omogo, Collins O.; Adung'a, Vincent O.; Krüger, Timothy; Masiga, Daniel K.; Engstler, Markus Species-Specific Adaptations of Trypanosome Morphology and Motility to the Mammalian Host African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate's incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the 'cellular waveform'. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites. 2016 e1005448 PLoS Pathogens 12 2 urn:nbn:de:bvb:20-opus-146513 10.1371/journal.ppat.1005448 Theodor-Boveri-Institut für Biowissenschaften OPUS4-14636 Wissenschaftlicher Artikel Dejung, Mario; Subota, Ines; Bucerius, Ferdinand; Dindar, Gülcin; Freiwald, Anja; Engstler, Markus; Boshart, Michael; Butter, Falk; Janzen, Chistian J. Quantitative proteomics uncovers novel factors involved in developmental differentiation of Trypanosoma brucei Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. 2016 e1005439 PLoS Pathogens 12 2 urn:nbn:de:bvb:20-opus-146362 10.1371/journal.ppat.1005439 Theodor-Boveri-Institut für Biowissenschaften OPUS4-13066 Wissenschaftlicher Artikel Weiße, Sebastian; Heddergott, Niko; Heydt, Matthias; Pflästerer, Daniel; Maier, Timo; Haraszti, Tamas; Grunze, Michael; Engstler, Markus; Rosenhahn, Axel A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming. 2012 e37296 PLoS One 7 5 urn:nbn:de:bvb:20-opus-130666 10.1371/journal.pone.0037296 Theodor-Boveri-Institut für Biowissenschaften OPUS4-11972 Wissenschaftlicher Artikel Batram, Christopher; Jones, Nivola G.; Janzen, Christian J.; Markert, Sebastian M.; Engstler, Markus Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness. 2014 eLife 3 e02324 urn:nbn:de:bvb:20-opus-119727 10.7554/eLife.02324 Theodor-Boveri-Institut für Biowissenschaften OPUS4-11534 Wissenschaftlicher Artikel Stellamanns, Eric; Uppaluri, Sravanti; Hochstetter, Axel; Heddergott, Niko; Engstler, Markus; Pfohl, Thomas Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation. 2014 Scientific Reports 4 6515 urn:nbn:de:bvb:20-opus-115348 10.1038/srep06515 Theodor-Boveri-Institut für Biowissenschaften OPUS4-6666 Wissenschaftlicher Artikel Heddergott, Nico; Krüger, Timothy; Babu, Sujin B.; Wei, Ai; Stellamanns, Erik; Uppaluri, Sravanti; Pfohl, Thomas; Stark, Holger; Engstler, Markus Trypanosome Motion Represents an Adaptation to the Crowded Environment ofthe Vertebrate Bloodstream Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy 2012 urn:nbn:de:bvb:20-opus-78421 Theodor-Boveri-Institut für Biowissenschaften