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

The control of monoallelic expression, antigenic variation and development of Trypanosoma brucei

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.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

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

A quorum sensing-independent path to stumpy development in <i>Trypanosoma brucei</i>

<div><p>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.</p></div

ES-attenuation induces stumpy development independent of cell density and SIF.

<p>The possible effects of SIF on a growth arrested clone of the GFP:PAD_UTR cell line were determined. Since SIF accumulates in the cell culture medium and induces stumpy development in a cell density-dependent manner, trypanosomes were induced for ectopic VSG overexpression at two different starting cell densities: (A) High parasite density (HD, 2.5x 10⁵ cells/ml) and B) low parasite density (LD, 2.5x 10⁴ cells/ml). The parasites were cultivated without dilution to allow the accumulation of SIF. Cumulative growth curves were recorded (+tet) for 9 days to analyze the possible impact of SIF on the growth arrested ectopic VSG overexpressor. To determine at which time point the ES-attenuation escapers could resume growth, the culture medium was exchanged after either 1 or 2 days of induction by washing (washed at day 1 +tet or 2 +tet). Induction was maintained by re-addition of tetracycline, and cultures were diluted again once they resumed growth. Data are means (± SD) of three experiments. Due to the small standard deviation the error bars are not visible. For visualisation of the actual cell densities and standard deviations non-cumulative growth curves, including non-induced cells and the parental AnTat1.1 cell line as controls, are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s013" target="_blank">S13 Fig</a>.</p

Overexpression of an ectopic VSG causes distinct growth phenotypes.

<p>(A) A reporter cell line with a GFP open reading frame integrated into the promotor region of the active AnTat1.1 ES (GFP^ESproA1.1^ES) was generated. The resulting trypanosome clones displayed a homogenous cytoplasmic GFP-signal (green). Nuclear (N) and mitochondrial DNA (K) were stained with DAPI (white). Scale bar: 5 μm. (B) A stumpy reporter cell line with a GFP:PAD1_UTR construct integrated into the tubulin locus was generated (GFP:PAD1_UTRA1.1^ES). The transgenic trypanosomes were adjusted to 5x 10⁵ cells/ml and cultivated for two days without dilution. As a consequence of cell density induced quorum sensing, the reporter cell line expressed the stumpy GFP-reporter in the nucleus (green) and the endogenous surface protein PAD1 on the plasma membrane (anti-PAD1 antibody; magenta). Scale bar: 5 μm. (C) Transfection of the ES-promoter reporter line (GFP^ESproA1.1^ES) with the inducible VSG 121 overexpression construct (121^tet) yielded the GFP^ESproA1.1^ES121^tet cell lines, while (D) transfection of the stumpy reporter cell line with 121^tet yielded the GFP:PAD1_UTRA1.1^ES121^tet cell lines. (C, D) After induction of VSG121 overexpression clonal populations of both reporter cell lines revealed different growth phenotypes. The trypanosomes either continued (proliferating) or ceased growth (arrested). Only the arrested clones expressed the GFP:PAD1_UTR stumpy reporter (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s008" target="_blank">S8 Fig</a>). Representative growth curves of tetracycline-induced (triangles) and non-induced cells (squares) of proliferating and growth arrested clones are shown. Each graph represents one clone and the data are means (± SD) of three experiments. Cumulative growth curves, including one of the parental AnTat1.1 cell line as a control, are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s002" target="_blank">S2 Fig</a>.</p

VSG silencing without ES-attenuation is not sufficient to trigger stumpy differentiation.

<p>The experiments were conducted with a proliferating clone of the GFP:PAD1_UTR reporter cell line at densities below 5x 10⁵ cells/ml (except the density-induced control). (A) The cell cycle position of DAPI-stained trypanosomes was analyzed after 24 and 48 hours of tetracycline induction. Non-induced slender (0 h) and density-induced stumpy cells (st) served as controls. Values are given as percentages (± SD) of two experiments (total n > 700). (B) On the left DAPI staining (grey) illustrates different dividing stages (indicated by yellow arrowheads). The green GFP:PAD1_UTR stumpy marker signal is absent. The DIC image on the right illustrates the typical slender morphology of proliferating ectopic VSG overexpressors. Note the characteristic extended free part of the flagellum (white arrowhead). Scale bar: 10 μm. (C) Western blot stained with an antibody against the mitochondrial lipoamide dehydrogenase (LipDH, green), whose expression increases during stumpy development. This reveals the uniformly low LipDH expression in proliferating ectopic VSG overexpressors during the time course of induction. Detection of paraflagellar rod (PFR) proteins served as a loading control (magenta).</p

Overexpression of an ectopic VSG causes silencing of the ES-resident VSG.

<p>Both (A, B) mRNA and (C, D) protein levels of the endogenous VSG A1.1 (green) and the ectopic VSG 121 (magenta) were monitored in growth arrested (left) and proliferating (right) clones during the course of ectopic VSG overexpression. In all graphs triangles correspond to the ES-promoter cell line (GFP^ESproA1.1^ES121^tet) and circles to the stumpy reporter cell line (GFP:PAD1_UTRA1.1^ES121^tet). Note that the initial VSG overexpression levels are comparable in arrested and proliferating parasites. For the quantification of (A, B) mRNA levels, total RNA samples were dot-blotted and hybridized with infrared fluorescently labeled probes, specific for <i>VSG 121</i> or <i>VSG A1</i>.<i>1</i>. The data were quantified and normalized to <i>β-tubulin</i> mRNA using the Licor Odyssey system. (C, D) VSG protein levels were quantified by dot-blotting 6x 10⁵ cell equivalents. The blots were incubated with an anti-VSG 121 or an anti-VSG A1.1 antibody. A histone H3 antibody was used for normalization. The VSG expression levels are given relative to VSG 121 expression levels of MITat1.6 wild type cells and parental AnTat1.1 cells natively expressing VSG A1.1. The dashed grey line indicates wild type expression levels (100%).</p

Overexpression of an ectopic VSG causes surface coat exchange.

<p>Immunostaining of a proliferating GFP:PAD1_UTRA1.1^ES121^tet clone using antibodies against the ectopic VSG 121 (magenta, left) and the endogenous VSG A1.1 (green, middle). MITat1.6 wild type cells natively expressing VSG 121 and AnTat1.1 wild type cells natively expressing VSG A1.1 were used as controls for antibody specificity. Non-induced cells (0) and VSG overexpressing parasites induced for 24 hours were analyzed. The merged images are shown in the right panel. DNA stained with DAPI (grey) is displayed in the merged image only. Scale bar: 20 μm. Flow cytometry quantifications of parasites stained with an antibody against the ectopic VSG 121 are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s003" target="_blank">S3 Fig</a>.</p

ES-attenuation-induced stumpy trypanosomes possess full developmental competence for tsetse transmission.

<p>(A) Flow cytometric analysis of the expression of the procyclic surface protein EP1. ES-attenuation-induced stumpy cells ectopically overexpressing VSG 121 for 48 hours were treated with CCA (3mM <i>cis</i>-aconitate and 3mM citrate at 27°C). Non-induced long slender cells (0 h) served as a negative and density-induced stumpy cells (st) as a positive control. After 0, 6 and 24 hours of CCA treatment, the trypanosomes were chemically fixed and immunostained for the detection of EP1. Cells in the M1 region of the plots are EP1-positive. (B) Representative immunofluorescence image of ES-attenuation-induced stumpy cells after 24 hours of CCA treatment. EP1 (magenta) is uniformly distributed on the surface, and the parasites show the characteristic shape of procyclic trypanosomes. Note the elongated posterior pole of the cells (white arrowhead). DAPI staining (grey) confirms the rearrangement of kinetoplast/nucleus and the re-entry into the cell cycle. Scale bar: 5 μm. (C) ES-attenuation-induced stumpy trypanosomes can passage through the tsetse fly. Flies were dissected at the earliest 50 days after infection. Typical parasite stages were present in different infected organs as illustrated by the shape of the cells (DIC images, upper panel). Staining of the flagellum with an antibody against PFR (magenta) shows the characteristic changes in flagellar length. DAPI staining (grey) illustrates the repositioning of kinetoplast and nucleus during developmental progression (lower panel). Scale bar: 5 μm.</p