TY - JOUR A1 - Zimmermann, Henriette A1 - Subota, Ines A1 - Batram, Christopher A1 - Kramer, Susanne A1 - Janzen, Christian J. A1 - Jones, Nicola G. A1 - Engstler, Markus T1 - A quorum sensing-independent path to stumpy development in Trypanosoma brucei JF - PLoS Pathogens N2 - 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. KW - Trypanosoma KW - hyperexpression techniques KW - parasitic cell cycles KW - cloning KW - cell cycle and cell division KW - cell differentiation KW - tetracyclines KW - parasitic diseases Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158230 VL - 13 IS - 4 ER - TY - JOUR A1 - Hartel, Andreas J.W. A1 - Glogger, Marius A1 - Jones, Nicola G. A1 - Abuillan, Wasim A1 - Batram, Christopher A1 - Hermann, Anne A1 - Fenz, Susanne F. A1 - Tanaka, Motomu A1 - Engstler, Markus T1 - N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold JF - Nature Communications N2 - 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. KW - parasitology KW - cellular imaging KW - membrane biophysics KW - single-molecule biophysics Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171368 VL - 7 ER - TY - JOUR A1 - Batram, Christopher A1 - Jones, Nivola G. A1 - Janzen, Christian J. A1 - Markert, Sebastian M. A1 - Engstler, Markus T1 - Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei JF - eLife N2 - 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. KW - antigenic variation KW - expression site attenuation KW - developmental reprogramming KW - cell biology KW - genes and chromosomes KW - Trypanosoma brucei KW - variant surface glycoprotein (VSG) KW - monoallelic expression Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119727 SN - 2050-084X VL - 3 IS - e02324 ER - TY - THES A1 - Batram, Christopher T1 - Die Kontrolle der monoallelen Expression, antigenen Variation und Entwicklung in Trypanosoma brucei T1 - The control of monoallelic expression, antigenic variation and development of Trypanosoma brucei N2 - Die ausschließliche Expression von nur einem Gen aus einer großen Genfamilie ist ein weit verbreitetes Phänomen, das als monoallele Expression bezeichnet wird. In dem Blutparasiten Trypanosoma brucei stellt die Expression eines einzigen variablen Oberflächenglykoproteins (VSG) aus einem Repertoire von über 1000 verschiedenen Genen die Grundlage für die Immunevasion dar. Durch einen periodischen Wechsel der VSG Expression (Antigene Variation) bleibt der Parasit vom Immunsystem des Wirtes unerkannt. Die VSG Gene werden aus telomerischen Blutstromform Expressionsstellen (BES) transkribiert, von denen nur eine zu einem bestimmten Zeitpunkt aktiv ist. Die Kontrolle der monoallelen VSG Expression ist somit einer der wichtigsten Virulenzfaktoren von T. brucei. Ziel dieser Arbeit war es, die Vorgänge eines transkriptionellen Wechsels zwischen zwei BESs zu beschreiben. Das Ausschalten des aktiven VSGs durch RNA-Interferenz hatte zuvor gezeigt, dass dies nicht zu einer erhöhten Wechselrate führt. Es wurde daher untersucht, welche Auswirkungen das Anschalten einer zweiten BES auf die monoallele Expression hat. Da es bisher keine Möglichkeit gibt, eine inaktive BES gezielt zu aktivieren, wurde ein artifizielles System gewählt, das die gezielte induzierbare Expression eines Gens ermöglicht. Die BESs unterscheiden sich in der Anzahl und Zusammensetzung der Expressionsstellen-assoziierte-Gene (ESAGs), jedoch besitzt jede BES ein telomernahes VSG. Somit wird, bei einer BES Aktivierung, in jedem Fall ein neues VSG exprimiert. Durch die induzierbare Expression eines zweiten VSGs wurde so das Anschalten einer neuen BES simuliert. Mithilfe dieses Systems konnte gezeigt werden, dass das VSG selbst für die Kontrolle der monoallelen Expression verantwortlich ist. Die ektopische Überexpression eines zweiten VSGs führte zu einer graduellen Inaktivierung der BES. Infolge dessen verlangsamte sich der Zellzyklus und die Zellen verblieben bis zu fünf Tage in einem ruhenden Zustand. Genauere Analysen dieses Zustandes zeigten, dass es sich hierbei um ein bisher unbekanntes, reversibles Zwischenstadium zwischen proliferierenden sogenannten Long Slender und arretierten sogenannten Short Stumpy Formen handelt. Die Ergebnisse dieser Arbeit führten zu einem neuen Modell, das die Kontrolle der monoallelen VSG Expression mit der Entwicklung der Trypanosomen mechanistisch verbindet. N2 - The exclusive expression of only one gene from a gene family is a common phenomenon, known as monoallelic expression. The blood parasite Trypanosoma brucei evades the host immune system by expressing only one variant surface glycoprotein (VSG) from a repertoire of hundreds of different VSG genes. By periodically switching VSG expression (antigenic variation) the parasites evade the host antibody response. The VSG genes are transcribed from specialized telomeric bloodstream form expression sites (BESs), of which only one is active at any given time. Thus, monoallelic VSG expression is one of T. brucei's most important virulence factors. The aim of this work was to describe the processes occuring while transcription switches from one BES to another. The depletion of the active VSG by RNA interference (RNAi) was shown previously to have no effect on switching frequency. It was therefore investigated here, which influence the activation of a new BES would have on monoallelic expression. So far, it has not been possible to specifically activate a silent BES. Therefore, an artificial system was chosen which allows for inducible expression of a particular gene. The BESs differ in number and composition of expression site associated genes (ESAGs), but all contain a telomeric VSG gene. Thus, activation of a new BES will inevitably lead to expression of a second VSG. To simulate - in the most straightforward manner - the activation of a new BES, a second VSG was inducibly expressed. Using this system, it was shown that the VSG itself controls its own monoallelic expression. The ectopic overexpression of a second VSG led to a gradual inactivation of the BES. This, in turn, led to a prolonged cell division cycle and the cells remained in a dormant stage for up to 5 days. Further analyzes of this stage revealed a new, reversible intermediate stage between proliferating long slender and arrested short stumpy forms. The results of this work led to a new model that mechanistically links the control of monoallelic VSG expression and development in trypanosomes. KW - Trypanosoma brucei KW - VSG KW - antigene Variation KW - monoallele Expression KW - Genexpression Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-90037 ER -