Fig 1.
Treponema pallidum exhibits a dynamic interaction with human platelets.
Darkfield video microscopy panels at 1000x show a treponeme attached to a fully activated platelet by both tips (green arrows 43.21 s). This treponeme alternated between coiling with vigorous axial rotation (2.68 Hz) against the platelet membrane (cyan curved arrow indicates the direction of rotation) (1.68–10.07 s, 28.77 s) and extending above the platelet (15.15 s, 21.12 s, 34.40 s), then remained attached by both tips (43.21 s) for a further 18 min 23 s of observation. Scale bar = 10 μm.
Fig 2.
Treponema pallidum-platelet interactions.
(A) Flow cytometry isolates the co-localized platelet and treponeme populations by SSC and FSC gating. Dot plots of (B) CFSE-labeled heat-treated treponemes demonstrate reduced binding to human platelets labeled with PE/Cy5 anti-CD41a compared with (C) CSFE-labeled viable treponemes. (D) CFSE-labeled viable treponemes bound significantly more human platelets (mean = 55.13% ± 2.91 [SD] *P<0.0001) compared with heat-treated treponemes (mean = 19.05% ± 2.29 [SD]) following co-incubation for 16 hours at 37˚C and ~ 5% oxygen. Results represent the mean of three independent experiments with statistical significance computed by two-way ANOVA, with a minimum of 3 replicates per sample type per experiment. (E) Darkfield microscopy FOV counts (20 random locations/slide) demonstrate viable treponemes bind significantly more human platelets (mean = 31.73% ± 1.19 [SD] *P<0.0001) than heat-treated treponemes (mean = 6.47% ± 1.19 [SD]) following co-incubation at 5% oxygen at 34˚C for 48 hours. Results represent the mean of three independent experiments with statistical significance computed by unpaired two-tailed Student’s t test, n = 3. (F) Heat-treated treponemes (top) are non-motile yet morphologically similar to viable treponemes (bottom). (G) Darkfield microscopy at 1000x magnification demonstrates platelet interactions are reversible. Image capture from video micrographs show edgewise attachment of a treponeme to an activated platelet (0.01 s to 8.10 s) which then detaches and moves away (9.05 s).
Fig 3.
Binding of T. pallidum to platelets increases with platelet activation.
(A) Treponemes displayed preferential binding to fully activated spread platelets (blue), with 57.8% of viewed images showing an interaction between fully activated platelets and T. pallidum. Treponemes bound to activated spheroid platelets (lime) in 37.6% of images, while platelets in early activation (yellow) constituted only 4.8% of the observed interactions. Treponema pallidum was never observed to bind to inactive platelets in 125 interactions analyzed from 75 images containing a total of 422 platelets (S1 Table). Interactions and activation states were observed at 1000x magnification using darkfield microscopy. (B) Images of platelets at different stages of activation at 1000x magnification using darkfield microscopy. Inactive platelets circulate as disks (grey) and bud pseudopods during early activation (yellow). Activated platelets may be spheroid with extended pseudopods (lime) or fully activated with an enlarged surface area and a very thin spread form (blue).
Fig 4.
Treponemes bind in multiple ways to activated platelets.
(A) Image analysis of 227 interactions in 158 micrographs demonstrated that 69% of treponemes bind platelets using one or both tips (dark blue). Both edgewise binding along the axial plane of the cell body (medium blue) and indirect binding via a platelet-bound treponeme or platelet pseudopod (lime) occurred in 14% of interactions, followed by “dynamic” binding (yellow), which occurred in 3% of interactions. (B) Treponemes interact with one or both tips (left), edgewise (middle) or indirectly via another treponeme (right). (C) Image capture of two treponemes with an activated platelet. Coiling of the treponeme on the platelet (0.79, 14.33, 16.21, 20.16, 21.47 and 24.41 s) with intervening periods of translation and extension away from the platelet by one (2.77, 20.16, 21.47 and 28.60 s) or both (6.70 s) treponemes was observed. White arrowheads indicate fully activated platelets. Scale bars = 5 μm.
Fig 5.
Motility parameters associated with Treponema pallidum-platelet interactions.
The movement and cell characteristics were contrasted between platelet-interacting (shaded circles) and non-interacting (open circles) treponemes utilizing high-resolution darkfield videos at 1000x magnification. (A) Treponeme wavelength (green bracket) and amplitude (yellow bracket) were measured only when the cell body waveform was in the plane of focus (grey bracket). Two activated platelets (white arrowheads) are bound by one treponeme. The grey bracket shows a segment of the treponeme with cell peak and troughs in the same focal plane. (B) Treponeme length was measured when the full treponeme was in the same focal plane (magenta bracket). (C) Platelet-interacting treponemes demonstrated a higher translational velocity (mean = 1.68 ± 0.10 μm/sec [SEM] *P < 0.0001, n = 24) compared with non-interacting treponemes (mean = 0.65 ± 0.10 μm/sec [SEM], n = 12). (D) Frame by frame analysis determined platelet-interacting treponemes rotate at a higher frequency (mean = 2.60 ± 0.27 Hz [SEM] **P = 0.0060, n = 12) versus non-interacting treponemes (mean = 1.46 ± 0.25 Hz [SEM], n = 11). (E) Top panel: Platelet-interacting treponemes maintained a more tightly coiled helix resulting in a shorter wavelength (mean = 0.79 ± 0.02 μm [SEM] ***P = 0.0237) versus non-interacting treponemes (mean = 0.86 ± 0.02 μm [SEM]), n = 15. Middle panel: Platelet-interacting treponemes demonstrated a trend towards a higher amplitude (mean = 0.30 ± 0.02 μm [SEM]) compared with non-interacting treponemes (mean = 0.27 ± 0.01 μm [SEM]), n = 15. Bottom panel: Platelet-interacting treponemes also displayed a trend towards a shorter, more compact coiled shape (mean cell length = 9.56 ± 0.65 μm [SEM]) compared with non-interacting treponemes (mean cell length = 10.46 ± 0.66 μm [SEM]), n = 15. Student’s unpaired two-tailed t tests were used to calculate statistical significance. Scale bars = 5 μm.
Fig 6.
Platelet-tethered treponemes experience reduced displacement and a lower speed than non-interacting treponemes.
An overlay of several video microscopy frames demonstrates the reduced speed and displacement experienced by a treponeme (green) that interacted with a slide-adhered platelet (asterisk). This interaction reduced its overall speed to 3.64 μm/sec and displacement to 48.64 μm (green dotted line) compared with a treponeme that did not interact (cyan) which has a speed of 8.73 μm/sec and displacement of 82.98 μm (cyan dotted line). Treponemes engaged in stationary adhesion (magenta arrowheads) maintained attachment when slide movement induced plasma movement. White arrow indicates the direction of flow. Scale bar = 5 μm.
Fig 7.
Treponema pallidum activates human platelets.
The potential for treponemes to activate platelets was assessed by flow cytometry (A) and using a plate-based assay (B). (A) Following 18 hours co-incubation at 37°C the results of two independent flow cytometry assays quantified platelet activation by the median fluorescence intensity (MFI) associated with CD41a receptor up-regulation. Platelets co-incubated with treponemes (hatched bar) demonstrated a significantly higher MFI (mean = 806.0 ± 37.76 [SEM], *P = 0.0118, n = 25) compared to resting platelets (white bar) (mean = 545.5 ± 104.4 [SEM], n = 18). There was no significant difference between treponeme co-incubated platelets and agonist pre-activated platelets (black bar) (mean = 746.5 ± 124.3 [SEM], n = 18). (B) Following co-incubation for 18 hours at 34°C the results of two independent platelet activation experiments were assessed by measuring the absorbance at 600 nm as a surrogate measure of fibrin clot formation. Platelets co-incubated with T. pallidum (hatched bar) had a higher absorbance (mean = 0.4137 ± 0.007 [SEM], *P = 0.0221, n = 20) compared to resting platelets (white bar) (mean = 0.3730 ± 0.018 [SEM], n = 12). There was no significant difference between treponeme-co-incubated platelets and activated platelets (black bar) (mean = 0.3791 ± 0.027 [SEM], n = 7).