Exosome secretion affects social motility in <i>Trypanosoma brucei</i>

<div><p>Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite <i>Trypanosoma brucei</i>, exosome secretion is induced by stress that affects <i>trans</i>-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, <i>Vps36</i>, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.</p></div

Exosome secretion affects social motility in <i>Trypanosoma brucei</i> - Fig 5

<p><b>(A) Immunogold TEM analysis demonstrating the location ZC3H41 in ILVs within MVBs.</b> Cells carrying the <i>SmD1</i> silencing construct, and the YFP-VPS36 construct were used. <b>(a, b)</b> un-induced (-Tet) or <b>(c-g)</b> silenced for 40 hrs. (+Tet) were used to prepare Cryo-EM sections, and were subjected to immunogold analysis. Imaging was performed using antibody to ZC3H41. <b>(B)</b> as in <b>(A)</b>, but using anti-GFP antibody to detect the VPS36. The scale bars are indicated.</p

Identification of SL RNA associated proteins.

<p><b>(A) Identification of proteins purified by affinity-selection</b>. Cells containing the <i>SmD1</i> silencing construct were induced for 48h, as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.ref009" target="_blank">9</a>]. An extract was prepared from 2×10⁹ cells. The extract was separated on a Superdex 200 column, and SL RNA containing fractions were subjected to affinity selection as described in Materials and Methods. Proteins obtained from the control experiment lacking the selecting oligonucleotide (-Oligo) and proteins from the affinity selected particles (+Oligo) were extracted from the streptavidin beads, separated on a 12% acrylamide SDS gel, and stained with silver. <b>(B) <i>ZC3H41</i>silencing.</b> Cell lines expressing the <i>ZC3H41</i> stem-loop silencing construct were induced for 48 hrs. Cells (~10⁶ cells/ lane) were subjected to western analysis using ZC3H41 and PTB1 antibodies. <b>(C) <i>ZC3H41</i> is an essential gene for trypanosome survival.</b> Cells were either induced (+Tet) or un-induced (-Tet), and growth was monitored. The arrow indicates the time of tetracycline addition. The number of un-induced cells is designated by triangles, and of induced cells by squares. <b>(D) ZC3H41 binds loosely to SL RNA.</b> Cells expressing TAP-Myc-His- ZC3H41 fusion protein and the <i>SmD1</i>-silencing construct were silenced for 48 hrs. Cells (1.5×10⁹) were UV irradiated, as described in Materials and Methods. Extracts prepared from control (-UV) cells, and cells following UV irradiation were affinity selected on IgG beads. The RNA was extracted from the beads and analyzed by primer extension with SL and U3 RNAs specific primers. T, Total extract (5%); S, supernatant after removing the IgG beads (5%); P, the entire RNA sample bound to beads. The position of the cap-4 modification is indicated. <b>(E) Depletion of ZC3H41 in <i>SmE/ ZC3H41</i> silenced cells.</b> Western analysis was performed, as described in panel B. <b>(F) Levels of SL RNA under <i>SmE</i> and <i>SmE/ ZC3H41</i> silencing.</b> 10 μg of total RNA was subjected to primer extension with primers specific to SL RNA, U4, and U3 snoRNAs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.s011" target="_blank">S1 Table</a>). The extension products were separated on a 6% denaturing gel. The identity of the cell line and the position of the modified cap nts are indicated. The statistical analysis represents the mean ± s.e.m of quantification from three independent experiments. **<i>P</i> <0.01, and ***<i>P</i> <0.005 compared to–Tet, using Student's <i>t</i>-test. <b>(G) Changes in localization of ZC3H41 and SL RNA during <i>SmD1</i> silencing.</b> Cells carrying the <i>SmD1</i> silencing construct were induced for the time indicated and subjected to <i>in situ</i> hybridization with SL RNA (red) and IFA with ZC3H41 antibodies (green). The nucleus was stained with DAPI. The merge was performed on DAPI staining and SL RNA hybridization and the time points of silencing are indicated.</p

Exosome secretion repels the migration of wild-type cells.

<p><b>Cells (~10</b>^<b>7</b><b>) were plated on semi solid agar containing tetracycline at a distance of 2.5 cm from each other.</b> The <i>SmD1 and SmD1/Vps36</i> silenced cells were plated on plates containing tetracycline. Pattern formation was analyzed 2 days after plating. The parasites from the plates were blotted, and the blot was stained with Ponceau and then reacted with anti-GPEET and anti-EP. The distance between the wild-type colony and the adjacent colony is presented. The statistical analysis represents the mean ± s.e.m **P< 0.01 comparing the distance between wild-type to wild-type, wild-type to <i>SmD1and</i> wild-type to <i>SmD1/Vps36</i> silenced cells based on three independent experiments.</p

The SL RNA exosomes are distinct from stress granules and nanotubes.

<p>Exosomes were prepared from <i>SmD1</i>-silenced cells grown in medium containing FCS free of bovine exosomes, after 2 days of silencing. The exosomes were fractionated on a 15–50% sucrose gradient at 39,900 rpm in a SW41 rotor for 20 hr. Fractions (500 μl) were subjected to SEM and western analyses as well as to primer extension to monitor SL RNA. <b>(A) Fractionation of vesicles on the sucrose gradient.</b> The density of the fractions are given and SEM images derived from fractions 13–14 and fractions 17–18 are presented. <b>(B) Western analysis.</b> Fractions 10–21 were subjected to western analysis using the indicated antibodies. <b>(C) The location of SL RNA</b>. RNA was extracted from the different fractions and subjected to primer extension.</p

Entry of exosomes to target cells.

<p><b>(A) Fluorescence of exosomes stained with DiL and containing the ZC3H41-GFP.</b> Exosomes (20μl, representing 1/10 of the exosome preparation from 10⁹ cells) were incubated at room-temperature with 1:1000 dilution of the DiL and were analyzed by ImageStream. <b>(a)</b> The number of particles versus their area is presented. The peak corresponding to exosomes is indicated. <b>(b) The overlap between the two fluorescent dyes. (c)</b> Single exosome fluorescence. The fluorescence of GFP and DiL was recorded in addition to the overlay. DIC, and the different fluorophores are indicated. <b>(B) Exosomes enter wild-type trypanosome cells. (a)</b> The exosomes described in <b>(A)</b> (40 μl) were incubated with 100μl containing 10⁶ cells. ImageStream analysis was performed after 10 minutes, and single-cell fluorescence of ZC3H41–GFP and DiL was monitored. <b>(b)</b> The level of internalization of the two fluorophores. <b>(C) Live imaging of cells incubated with ZC3H41-GFP exosomes and lysotracker.</b> Exosomes were prepared from 10⁹ cells, and 1/10 of the preparation (20 μl) was incubated with 100μl containing 10⁶ cells for 1 hr and then lysotracker was added, and cells were visualized.</p

Exosomes secreted from <i>SmD1</i> silenced cells.

<p><b>(A) TEM analysis of the exosome-enriched fraction from <i>SmD1</i> silenced cells.</b> Exosomes were purified, from 10⁹ cells after 40 hrs of silencing, and were subjected to TEM. <b>(a)</b> The entire field of exosomes; <b>(b)</b> and <b>(c)</b> show enlargement of a section indicated by a square on panel <b>(a). Exosome size distribution.</b> For the analysis, the size of 150 exosomes was determined from ten different images using ‘NIS-elements’ imaging Software (Nikon, USA). The data were analyzed by the one-sample Kolmogorov-Smirnov test (SPSS IBM, USA). The average size of each exosome was 106.1 ± 26.6 nm (p value for normal distribution, 0.69). <b>(B) RNA is secreted via exosomes.</b> Cells containing the <i>SmD1</i> silencing construct were silenced for the indicated times (hours). RNA was prepared from the cells and from exosomes as described in Materials and Methods. The RNA was subjected to primer extension. The products were separated on a 6% acrylamide denaturing gel. The cap-4 nts are indicated. <b>(C) Proteins are secreted with the exosomes.</b> Proteins were extracted from exosomes prepared from 10⁹ cells and were subjected to western analysis with the indicated antibodies. Total cell extract (20 μg) was used to monitor the specificity of the antibodies. <b>(D) Immunogold-SEM detecting ZC3H41 protein on the surface of the parasite.</b> Un-induced cells containing the <i>SmD1</i> silencing construct (-Tet) and <i>SmD1</i> silenced cells at the indicated time points were subjected to immunogold staining with specific anti-ZC3H41 antibodies. Backscatter images are presented; scale bars are indicated.</p

TEM analysis of <i>SmD1</i> silenced cells.

<p>Cells before and after silencing of <i>SmD1</i> (40 hrs) were fixed, and ultra-thin sections were prepared and examined by TEM. The different ultra-structures are indicated. MVB, multivesicular bodies; ILV, intraluminal vesicles; M, mitochondrion; ER, Enodoplasmic reticulum; scale bar, 500 nm. (<b>A and B</b>) Un-induced cells (-Tet). (<b>C-G</b>) induced cells. Section showing MVB containing ILVs. <b>(C-D)</b> MVB located near the plasma membrane, and secreted exosome present on the cell surface. <b>(E-F)</b> EVs secreted from the cell membrane and flagella pocket. <b>(G)</b> Larger vesicles emerging from the flagellar membrane. The white arrow-heads indicate the ILVs and black arrow-heads the EVs.</p

Exosome secretion affects social motility in <i>Trypanosoma brucei</i> - Fig 11

<p><b>(A) Effect on wild-type migration by different cell type. (a) Late PCF cells do not express GPEET.</b> Cells were plated on semi solid agar at a distance of 2.5 cm from each other. The plates were subjected to western analysis with GPEET and EP antibodies. The identity of the cells and incubation time are indicated. <b>(b) Migration of cells following longer incubation on plates</b>. Cells were plated on semi solid agar containing tetracycline at a distance of 2.5 cm from each other. For longer time points, 8×10⁵ cells (in 5 μl) were used. Wild-type, <i>SmD1</i> and <i>SEC63</i> were silenced on plates for the time indicated. Statistical analysis represents the mean ± S.E.M **P< 0.01 comparing the distance between wild-type cells and the silenced cells (based on three biological replicates). <b>(B) Free exosomes but not FCS divert the migration of parasites.</b> Cells (~10⁷) were plated on semi solid agar containing tetracycline at a distance of 2.5 cm from each other. The plates were seeded with either a solution of FCS (5 μg/μl) or with semi-purified exosomes (5 μg/μl). The drops (5 μl) of FCS and of exosomes (5 μl of extract from 10⁹ in 200μl) were added at the same spot every 12 hrs. for 3 days; scale bar, 5 mm. Statistical analysis represents the mean ± S.E.M **P< 0.01 comparing the distance between wild-type cells and exosomes versus wild-type cells and FCS (based on three biological replicates). <b>(C) Damaged exosomes or protein lysates do not affect the migration of cells. (a)</b> Migration of wild-type cells was monitored on plates that were seeded with exosomes prepared from cells subjected to heat-shock (HSE) (5 μl, exosomes prepared from 10⁹ cells i.e. 25 μg), and 25 μg of lysate prepared from <i>SmD1</i> silenced cells; <b>(b)</b> the same as in <b>(a),</b> but the plate was seeded with 25 μg of lysate prepared from <i>SmD1</i> silenced cells, lysate from wild-type cells, and exosomes which were pre-treated with NP40. The exosomes (1/10 purified from 10⁹ cells) from <i>SmD1</i> silenced cells (25 μg) were mixed with NP40 to 0.05% and were incubated over night before seeding on the plates. The plates were incubated for the indicated times. The position to which the substances were seeded are marked with black spots. The experiment was repeated three times.</p

MTR4 regulates the level of U snRNA, but does not affect SL RNA, under <i>SmD1</i> silencing.

<p><b>(A) Primer extension of SL, U2 and U3 RNAs during <i>SmD1</i> silencing.</b> Total RNA (10 μg) was extracted from cells silenced for the indicated times, and subjected to primer extension. The extension products were separated on a 6% denaturing gel. <b>(B)</b> The same experiment as in <b>(A)</b> but using RNA extracted from <i>SmD1/Mtr4</i> silenced cells. <b>(C) SL RNA granules are not P bodie</b>s. ZC3H41 localization with respect to P-bodies labeled with DHH1. Cells carrying the <i>SmD1</i> silencing construct, and the YFP-DHH1 construct were silenced for 40 hrs. and stained for IFA using ZC3H41 and YFP antibodies (red and green respectively). The nucleus was stained with DAPI. <b>(D) <i>In situ</i> hybridization with SL RNA.</b> Cells carrying the <i>SmD1</i> silencing construct and expressing YFP-DHH1 were induced for 40 hrs. and subjected to <i>in situ</i> hybridization with SL RNA (red) and immunofluorescence using YFP antibody for YFP-DHH1 (green). The nucleus was stained with DAPI. <b>(E) SL RNA granules are not stress granules</b>. Cells were silenced for 40 hrs. and stained by IFA using PTB1 antibodies (green stain) and were subjected to <i>in situ</i> hybridization with SL RNA (red). The nucleus was stained with DAPI. <b>(F)</b> as in <b>(E)</b> but using antibodies to eIF4E-1. The merge was performed between DAPI staining, IFA and <i>in situ</i> hybridization.</p