<i>TbS</i>UMO-SIM binding assays.

<p><b>(A)</b> Illustration of the domain arrangement in SIMx4 and mutSIMx4 probes. <b>(B)</b> Coomassie Blue stained SDS-PAGE analysis of probe purifications. <i>E</i>. <i>coli</i> BL21 DE3 cells expressing SIMx4 or mutSIMx4 probes were lysed and cleared lysates were subjected to Ni⁺² chromatography. Inputs, In; Eluates, El. <b>(C)</b> <i>E</i>. <i>coli</i> BL21 DE3 cells expressing HA-<i>Tb</i>SUMO chains (Inputs) were incubated with the SIMx4 or the mutSIMx4 probes. Samples were pulled-down using Ni⁺²-Sepharose beads. Proteins were visualized by Western blot using anti-HA antibodies. Coomassie stained gels of the inputs are shown. <b>(D)</b> Cell free extract from <i>T</i>. <i>brucei</i> 427 PCF was incubated with SIMx4 or mutSIMx4 probes, which were subsequently purified by Ni⁺² chromatography. The capture of SUMOylated proteins was determined by Western blot analysis using anti-<i>Tb</i>SUMO antibodies, while anti-α-tubulin was used as the loading control for inputs.</p

Chromatin organization in <i>Tb</i>SUMO<i>all</i>KR mutants.

<p><b>(A)</b> Genomic DNA isolated from HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) or HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) strains was digested during 5 minutes with increasing units of MNase (0, 3 and 7 U). Samples were separated on 2% agarose gels and visualized under UV light after ethidium bromide staining. Degradation products corresponding to DNA that had been bound to mono-, di-, or tri-nucleosomes, as well as undigested DNA (undig.) are indicated.Bands were quantified with Image Studio software and statistical analysis was performed using Student's t test. <b>(B)</b> Fluorescent <i>in situ</i> hybridization was used for telomere labelling (Tel, red). Representative images are shown for HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) parasites. DNA was visualized using DAPI (blue). N, nucleus; K, kinetoplast DNA. Merged images are shown.</p

Biological classification of good SIM candidate proteins in <i>T</i>. <i>brucei</i>.

<p>Sequences were identified in the translated proteome using a combination of search methods (see text) and 50 out of 102 hits were classified into functional categories according to their genome annotation or protein domains detected by PFAM database (<a href="http://pfam.sanger.ac.uk/" target="_blank">http://pfam.sanger.ac.uk</a>). GeneID, protein description, total number of SIMs and a scheme of the domains (in scale) are shown. Location of the SIM within each sequence is shown in coloured dot shape: magenta, SIM-a; bourdeux, SIM-b and light gray, SIM-r. Length of the proteins (in residues) are also summarized.</p

SUMO chain mutant <i>T</i>. <i>brucei</i> procyclic parasites.

<p><b>(A)</b> Schematic representation of the SUMO variants expressed in PCF parasites. GG, diglycine motif essential for SUMO processing, activation and conjugation. <b>(B)</b> Growth of HisHA-<i>Tb</i>SUMO<i>all</i>KR strain (<i>Tb</i>SUMO<i>all</i>KR) compared to HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and wild-type parasites. Wild-type and transgenic parasites were cultured up to one month without observing significant differences in growth rate. <b>(C)</b> Conjugating capacity of HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) parasites. Whole-cell extracts were boiled in Laemmli sample buffer immediately after harvesting, separated in a 7.5%-12.5% discontinuous acrylamide gel (3,5x10⁷ cells/lane), and analysed by Western blot using anti-HA antibodies. An unspecific cross-reacting band of ∼50 kDa from anti-<i>Tc</i>SUMO antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193528#pone.0193528.ref022" target="_blank">22</a>] was used as loading control. <b>(D)</b> Immunofluorescence analysis of wild-type, HisHA-<i>Tb</i>SUMO (<i>Tb</i>SUMO) and HisHA-<i>Tb</i>SUMO<i>all</i>KR (<i>Tb</i>SUMO<i>all</i>KR) parasites. Nuclear and kinetoplast DNA were visualized by DAPI staining (blue). Representative images of anti-<i>Tb</i>SUMO (green), anti-HA (green) and <i>Tb</i>SUMO/anti-HA-DAPI merged images are shown.</p

<i>Tb</i>SUMO chain formation <i>in bacteria</i>.

<p><b>(A)</b><i>E</i>. <i>coli</i> BL21 DE3 cells were used to co-express the complete SUMOylation system using either a wild type <i>Tb</i>SUMO variant (<i>Tb</i>SUMO) or a mutant version with all lysine residues replaced by arginines (<i>Tb</i>SUMO<i>all</i>KR). Cleared cell lysates were subjected to Ni⁺² affinity chromatography and input (In) and eluates (El) were analyzed by 10% SDS-PAGE followed by Coomassie Blue staining. The positions of <i>Tb</i>SUMO monomer (SUMO), dimers (SUMO)₂ and trimers (SUMO)₃ are indicated. Note that the E2 enzyme (upper band of the doublet in the lane that corresponds to the eluate from <i>Tb</i>SUMO<i>all</i>KR strain) copurifies with SUMO (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193528#pone.0193528.s001" target="_blank">S1 Fig</a>). <b>(B)</b> Comparison of the three dimensional structure of <i>Tb</i>SUMO (2K8H, red) and human SUMO-2 (2N1W, green). Sequence alignment of N-terminal extensions reveals a potential conserved SUMOylation site in <i>Tb</i>SUMO K27 marked with an asterisk. <b>(C)</b> <i>In bacteria</i> SUMOylation assays were performed with different HA-tagged <i>Tb</i>SUMO variants. Cleared lysates were subjected to Ni⁺² affinity chromatography and analysed by Western blot using anti-HA antibodies. Note that in addition to SUMO polymers, the bands marked with asterisks could also correspond to SUMOylated forms of other components of <i>T</i>. <i>brucei</i> SUMOylation machinery, such as <i>Tb</i>E2 or <i>Tb</i>E1a (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193528#pone.0193528.s001" target="_blank">S1 Fig</a>).</p

Anti-VSG IgM antibody response.

Detection of IgM antibodies against VSG221 in the serum of mice infected with WT or SUMO chain mutant (SUMO allKR) parasites at the first peak of parasitemia by ELISA. Serum samples were diluted 1:5, and absorbance values (Abs) were measured at 450 nm. C+: positive control; C-: negative control. (TIF)</p

Generation of SUMO chain mutant monomorphic BSF parasites.

(A) Schematic representation of the generation of TbSUMO allKR parasites. (B) Conjugating ability of WT TbSUMO (WT) and SUMO allKR parasites. Parasites were boiled in Laemmli’s sample buffer immediately after harvesting. Proteins were separated by electrophoresis using a 10% SDS-poliacrylamide gel (3x107 cells/lane). SUMO conjugates were analyzed by Western blot using anti-TbSUMO antibodies and anti-PAB-C antibodies as loading control. (C) Growth curves for SUMO allKR and wild type (WT) parasites. WT and transgenic parasites were cultured up to one month without observing significant differences in growth rate. Doubling time was calculated by daily subculture back to 1 × 105/ml to maintain log-phase growth (n = 3). (D) Immunofluorescence (IF) analysis of WT and SUMO allKR BSF parasites. Nuclear and kinetoplast DNA were visualized by DAPI staining (blue). Representative images of anti-TbSUMO (green) and anti-TbSUMO-DAPI merged images are shown. For WT cells 63% of the nuclei showed a single HSF (n = 261) and for SUMO allKR cells this percentage was 37% (n = 318). Scale bar 5 μm.</p

Schematic diagram summarizing the main experimental findings.

Schematic diagram summarizing the main experimental findings.</p

Mice infections with SUMO chain mutant pleomorphic parasites.

BALB/c mice were infected intraperitoneally with 5000 WT or SUMO allKR parasites. (A) Time course of parasitemia in mice infected with WT (n = 3, black circles) or SUMO allKR parasites (n = 7, red squares). Results are shown as the means and their corresponding SEM (gray shaded area). (B) Mice survival was monitored daily and is shown by a Kaplan-Meier curve for infections with WT (n = 3, black circles) and SUMO allKR parasites (n = 7, red squares). (C) Nucleus and kinetoplast configuration was analyzed in methanol-fixed blood smears stained with Hoechst. (D) Representative images of methanol-fixed blood smears of infected mice prepared and stained with anti PAD1 antibodies (green) and Hoechst (blue). (E) Quantification of PAD1 stained parasites of (D). The bottom panel shows representative images of parasites expressing PAD1 in the surface (PAD1 positive), parasites that do not show any labelling (PAD1 negative) and parasites that express low levels of PAD1 mainly in vesicles (intermediate cells). For (C-E) parasites were collected from the first peak of parasitemia (at 6 dpi). Experiments were performed in triplicates (WT, n = 36; SUMO allKR, n = 62). Scale bar 5 μm. Statistical test: two-tailed paired t test (*P < 0.05; **P < 0.01; ***P < 0.001).</p

Evaluation of stumpy parasites <i>in vivo</i>.

Representative images of methanol-fixed blood smears of infected mice on the peak of parasitemia (at 5 dpi) stained with (A) Giemsa; (B) anti PAD1 antibodies (green) and DAPI (blue). Stumpy cells from pleomorphic parasites are shown as positive control (arrowhead). Scale bar 10 μm. (TIF)</p