Figure 1.
TSP1, a secreted protein, accumulates on large vesicles upon prolonged binding of T.vaginalis to Ects.
T. vaginalis transfected with hemagglutin (HA) tagged TSP1 (Tvag_019180, green) in the presence of host ectocervical cells (Ects) for 15 min (A) or 60 min (B). Large vesicular structures containing Tsp1 that accumulate after 60 min are marked by arrows. Nuclei are labeled blue by DAPI stain. (C) Western blot analyses of supernatant and pelleted fractions harvested from T. vaginalis after incubation at 37°C or 16°C for 2 hrs. The blot was reacted with anti-HA antibodies to detect HA-tagged Tsp1 (∼35 kDa), anti β-tubulin (∼50 kDa) and anti neomycin phophotransferase (∼30 kDa) antibodies. Incubation at 16°C and detection β-tubulin and neomycin phophotransferase serve as negative controls for cell lysis.
Figure 2.
Physical characterization of T. vaginalis exosomes.
(A) Cup-shaped vesicles ∼50–100 nm in diameter were observed using negative staining EM. (B) Immunoblot analyses of sucrose gradient fractions (F1–F7) containing Tsp1-HA tagged exosomes reacted with anti-HA antibodies. Densities are listed above the fractions and molecular weight markers (in kDa) are shown on the left. Expected size of Tsp1-HA is ∼34 kDa, the band at 72 kDa is likely a dimer. T.vaginalis vesicles have a density of ∼1.06–1.25 g/cm3. (C) Nanosight trace of purified vesicles. A mean diameter of 95 nm was measured. (D) Table from Nanosight analysis of percentage of purified vesicles in various size ranges.
Figure 3.
(A) Mock gel of cellular and exosomal RNA analyzed using an Agilent 2100 Bioanalyzer. Molecular weight markers (in nucleotides (nts)) are indicated on the left. (B) Traces with the amounts of cellular or exosomal RNAs relative to size in nts are shown.
Figure 4.
(A) Silver stained polyacrylamide gel of whole cell lysate (WCL) and isolated exosomes, normalized by protein concentration. (B) The predicted function of the 215 proteins identified in exosomes isolated from strain B7RC2. (C) Table of percentage of exosomal proteins with transmembrane domain or signal peptides as annotated in TrichDB (www.trichdb.org) as well as percentage of exosomal proteins with orthologs in mammalian exosomes (ExoCarta database), Leishmania exosomes [37], or T.vaginalis surface proteome [15].
Figure 5.
BODIPY-PC labeled exosomal membranes transfer the label to Ects.
(A) BODIPY-PC labeled exosomes or BODIPY-PC labeled hydrogenosomes were incubated with Ects. After extensive washing, cells were DAPI-stained & viewed by fluorescence microscopy. (B) Ects transiently transfected with GFPS1-10 were incubated for 1 hr with exosomes from T. vaginalis exosomes expressing GFPS11 tagged proteins. Ects were washed, fixed, DAPI-stained & viewed by fluorescent microscopy. Incubation with exosomes containing the Tvag_180840 protein tagged with the GFPS11 fragment is shown; similar data was obtained using a second (Tvag_137880) exosomal protein fused with the GFPS11 fragment. (C) Quantification of GFP fluorescence for a representative experiment, showing mean ± standard deviation. Approximately 35% of Ects were transfected using a control full length GFP (left) whereas ∼20–25% of Ects fluoresce when Ects are transfected with GFPS1-10 and incubated with either Tv_180840 exosomes (middle) or Tv_137880 exosomes (right).
Figure 6.
Exosomes induce Ect secretion of IL6 and IL8 and specifically prime the VEC IL8 response to parasites.
(A & B) Ects were incubated with either 9 ug BSA, 5×105 parasites, 9 µg exosomes for 6 hr and the supernatants were removed and assayed for IL6 or IL8 cytokines as indicated using ELISA assays. (C & D) Ects were preincubated for 12 hr with either PBS, 9 ug BSA, or 9 ug exosomes followed by the addition of 5×105 parasites for 6 hr. Supernatants were then assayed for either IL6 or IL8 cytokines as indicated and data were normalized as percent of cytokine secretion using 9 ug BSA without the addition of parasites. (E) Ects were incubated with 9 ug of either exosomes, BSA, hydrogenosomes, cytosol or supernatant from the last step of the exosome purification protocol, as indicated, for 6 hr. Supernatants were spun to remove debris and then assayed for IL8. Data was normalized as percent of IL8 secretion using 9 ug exosomes as 100%. Mean values ± standard deviation of a representative experiment is shown.
Figure 7.
Preincubation with exosomes of a highly adherent strain increases adherence of a poorly adherent strain to Ects.
Poorly adherent parasite strain G3, Ects or both, as indicated, were preincubated with exosomes from the highly adherent B7RC2 strain (A) or poorly adherent G3 strain (B) for 1 hr, followed by washing to remove exosomes. Adherence of G3 parasites to the Ects was then measured. BSA preincubation of Ects (indicated as BSA) served as a negative control and was used to normalize experiments. (C) B7RC2 (highly adherent) parasites, Ects, or both, as indicated, were preincubated with exosomes from B7RC2 for 1 hr, followed by washing, and determination of adherence. The mean of three experiments done in triplicate per experiment is shown ± SEM.
Figure 8.
Exosomes from highly adherent strains increase attachment of poorly adherent G3 parasites to host cells while exosomes from strains of equal or lesser adherence do not affect G3 attachment.
Poorly adherent G3 parasites, Ects or both, as indicated, were preincubated with exosomes from the T1, RU384, MSA1103, or LSU160 strain for 1 hr, followed by washing to remove exosomes. Adherence of G3 parasites to the VECs was then determined. The mean of 3–5 experiments with each treatment done in triplicate per experiment is shown ± SEM.
Figure 9.
Exosomes from strains more highly adherent to BPH1 cells than Ects have a greater effect on G3 attachment to BPH than to Ects.
Ect or BPH1 cells were preincubated with exosomes from either the G3, MSA1103, LSU160, or B7RC2 strain for 1 hr as indicated, followed by washing to remove exosomes. Adherence of G3 parasites to either BPH1 or Ects was measured. BSA preincubation of Ect or BPH1 cells was used to normalize the data. Mean of fold increase in attachment relative to BSA control of 3–5 experiments +/− SEM is shown.
Figure 10.
Visual summary of role of T.vaginalis exosomes.
T.vaginalis secretes exosomes that package core conserved exosomal proteins as well as those unique to the parasite that may be involved in pathogenesis. Contents of exosomes may be strain specific and vary in their ability to increase parasite attachment to host cells. Parasite exosomes can fuse with hose cells and deliver their contents. These contents modulate host immune response and potentially factors for parasite adhesion.