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

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

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

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

Proliferating and arrested clones respond to SIF with development to the stumpy stage.

<p>Slender parasites of a proliferating and an arrested clone of the GFP:PAD1_UTR reporter cell line were cultivated without dilution. This allowed the accumulation of the parasite secreted stumpy inducing factor (SIF). This was done in the absence of tetracycline, i.e. without VSG overexpression. Therefore, we here refer to potentially proliferating ('proliferating') and potentially arrested ('arrested'). (A) Representative growth curves of a ‘proliferating’ (squares) and an ‘arrested’ (circles) clone. The arrows indicate the time points at which the number of GFP:PAD1_UTR-positive cells was determined (see B). Control growth curves for the parental GFP:PAD1_UTR reporter cell line are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006324#ppat.1006324.s010" target="_blank">S10 Fig</a>. Data are means (± SD) of three experiments. (B) Quantification of cells expressing the GFP:PAD1_UTR reporter. The expression was monitored at cell densities of 5x 10⁵ cells/ml (0 h), as well as after 24 hours and 48 hours of growth. Values are percentages (± SD) of three experiments (total n > 600 cells).</p