The model trypanosome and a real trypanosome.

<p>(a) Cell body of the model trypanosome without distortion. The elastic network made from vertices connected by springs defines the surface. The blue line connecting a series of vertices represents the flagellum with the helical half-turn. (b, c) Snapshots of the model trypanosome during simulated swimming motion. (d) 3d volume model of a live trypanosome with fluorescently labeled surface. (e) 3d surface model of the cell in (d), with the flagellum highlighted in blue. (f) The same surface model rotated about the horizontal, in order to get a better view on the left-handed half-turn of the flagellum indicated in red.</p

Comparison of the model and a real trypanosome during swimming motion (A–D).

<p>The swimming trajectory and dynamic shape of the simulated model trypanosome (top row) compares well with the forward swimming motion of the real trypanosome (middle and bottom row). Snapshots of the real trypanosome are taken at the indicated times from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003967#pcbi.1003967.s005" target="_blank">S4 Video</a>. Fluorescently labelled surface (middle row) or untreated cells (bottom row) were recorded by high speed microscopy (200–500 frames per second). The cells, which exhibit similar speeds and rotational frequencies, show matching cell body conformations at all times over a swimming path of several cell lengths. These periodically repeating shape conformations are initiated by the bending wave passing along the flagellum and determine the trajectory of the swimming parasite.</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

ES attenuation accelerates SIF-induced stumpy formation at the population level.

<p>The GFP:PAD1_UTR cell line was challenged with ES-attenuation and chemical triggers for stumpy development. The trypanosome culture was adjusted to a cell density of 1x 10⁵ cells/ml and one part was induced with tetracycline (ES). Non-induced cells served as a control (no trigger) and were treated with 200μm cAMP or 0.25x SIF (cAMP, SIF). The last two cultures were tetracycline-induced additionally received the stumpy triggers (ES + cAMP, ES + SIF). After 20 hours, the numbers of GFP:PAD1_UTR-positive trypanosomes was counted. The values are means ± SD (n > 1200 cells each). ES-attenuation in combination with either SIF or cAMP produces more stumpy cells than the summed effect of the individual cues.</p

Outgrowing ectopic VSG 121 overexpressors are fully competent for stumpy differentiation and retain the tetracyclin-inducible gene expression system.

<p>The analyses were done with a growth arrested clone of the GFP:PAD1_UTR cell line that had resumed growth following tetracycline induction. (A) Representative cumulative growth curves of tetracycline-induced (triangles) and non-induced (squares) cells. The parental AnTat1.1 cell line (circles) served as a growth control. Data are means (± SD) of two experiments. Due to the small standard deviation the error bars are not visible. (B) After 8 days of induction, ectopic VSG overexpressors that had resumed growth (8 d tet) and non-induced cells (-tet) were treated with the stumpy-differentiation triggers pCPT-cAMP (200 μM) and SIF (0.25x). Control cells did not receive either compound (control). The number of GFP:PAD1_UTR positive cells was microscopically determined after 20 and 28 hours of treatment. Values are percentages (± SD) of experiments performed in triplicate (total n > 600 cells). (C, D) Ectopic VSG overexpression is re-inducible, but growth arrest is not. After 48 hours of ectopic VSG overexpression tetracycline was removed. The parasites were further cultivated without tetracycline for one week before re-addition of the antibiotic. (C) Trypanosomes cultivated without tetracycline for one week did not express the ectopic VSG 121 (magenta), but the ES-resident VSG A1.1 (green). When tetracycline was re-added, the parasites again expressed the ectopic VSG 121 and suppressed VSG A1.1. DNA was stained with DAPI (grey). Scale bar: 20 μm. (D) Cumulative growth curves of trypanosomes after removal (white squares) and re-addition of tetracycline (grey triangles). Tetracycline was removed 48 hours post-induction, and the cells were cultivated for 7 days in the absence of the drug. Then tetracycline was re-added (grey triangles) or not (squares) and population growth was determined. As a control, parasites of the same clone were induced with tetracycline for the first time (white triangles). 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 the standard deviation, the data are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s011" target="_blank">S11 Fig</a> as non-cumulative growth curves.</p

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

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

Silencing of the ES-resident VSG is independent of ES-attenuation.

<p>The transcriptional status of the active ES of growth arrested (left) and proliferating (right) ectopic VSG 121 overexpressors was monitored. (A, B) The GFP^ESpro reporter was used to detect transcripts from the ES promotor region (<i>GFP</i>) and (C, D) <i>ESAG6</i> was measured as an example for a native ES transcript. All data points reflect measurements on the single cell level using mRNA FISH (Affymetrix). The signal intensity in deconvolved, summed slice projections (100 images, z-step 100 nm) was measured with Image J and is represented as relative fluorescent unit (RFU). Two different induction times (24 and 48 h) were analyzed, and non-induced long slender (0 h) or density-induced short stumpy cells (st) served as controls. Only parasites in G1-phase of the cell cycle were analyzed. The magenta bars are means ± SD (n > 100). Statistical analysis was conducted using an unpaired t-test (not significant (n.s.) p-value > 0.05; ** p-value < 0.01; *** p-value < 0.001).</p

Ectopic VSG overexpression-induced ES-attenuation causes stumpy development.

<p>The analyses were conducted with an arrested clone of the GFP:PAD1_UTR reporter cell line at densities below 5x 10⁵ cells/ml (except for 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. The configuration of the kinetoplast (K) and nucleus (N) was microscopically analyzed. Only cells with a non-dividing kinetoplast and a single nucleus are in the G1-phase (1K1N). Parasites with a dividing kinetoplast (1K^d1N) are in the mitochondrial S-phase. Trypanosomes with two kinetoplasts (2K1N) are in G2/M-phase, while parasites with two kinetoplasts and nuclei (2K2N) are post mitotic. Parasites with an abnormal K/N configuration were termed "other". Values are given as percentages (± SD) of two experiments (total n > 500). (B) Representative microscopic image of cells ectopically overexpressing VSG 121 for 48 hours. In the left image DAPI (grey) and GFP:PAD1_UTR fluorescent signals (green) illustrate the 1K1N cell cycle arrest and the expression of the stumpy stage reporter. The DIC image on the right documents the short stumpy morphology of the trypanosomes. Note the characteristic short 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 shows that LipDH is up-regulated during ectopic VSG overexpression-induced ES-attenuation. Detection of paraflagellar rod (PFR) proteins served as a loading control (magenta). (D) Deconvolved three-channel 3D images of chemically fixed non-induced slender cells (0 h, upper panel) and parasites ectopically overexpressing VSG 121 for 48 hours (48 h, lower panel). The mitochondrion was stained with mitotracker (magenta), the GFP:PAD1_UTR reporter is shown in green and DAPI in grey. The upper panels show the shape of typical elongated mitochondria of slender cells, and the lower ones branched stumpy-like mitochondria of ectopic VSG overexpressors (48 h). Scale bars: 5 μm.</p