Identification of Tp0751 (Pallilysin) as a Treponema pallidum Vascular Adhesin by Heterologous Expression in the Lyme disease Spirochete

Abstract

Treponema pallidum subsp. pallidum, the causative agent of syphilis, is a highly invasive spirochete pathogen that uses the vasculature to disseminate throughout the body. Identification of bacterial factors promoting dissemination is crucial for syphilis vaccine development. An important step in dissemination is bacterial adhesion to blood vessel surfaces, a process mediated by bacterial proteins that can withstand forces imposed on adhesive bonds by blood flow (vascular adhesins). The study of T. pallidum vascular adhesins is hindered by the uncultivable nature of this pathogen. We overcame these limitations by expressing T. pallidum adhesin Tp0751 (pallilysin) in an adhesion-attenuated strain of the cultivable spirochete Borrelia burgdorferi. Under fluid shear stress representative of conditions in postcapillary venules, Tp0751 restored bacterial-vascular interactions to levels similar to those observed for infectious B. burgdorferi and a gain-of-function strain expressing B. burgdorferi vascular adhesin BBK32. The strength and stability of Tp0751- and BBK32-dependent endothelial interactions under physiological shear stress were similar, although the mechanisms stabilizing these interactions were distinct. Tp0751 expression also permitted bacteria to interact with postcapillary venules in live mice as effectively as BBK32-expressing strains. These results demonstrate that Tp0751 can function as a vascular adhesin.

Figure 1

Interaction of Tp0751-expressing strains with human endothelia under static and shear stress conditions. (a) Mean ± SEM numbers of parent and Tp0751-expressing B. burgdorferi (Bb-Tp0751) adhered to primary human umbilical vein endothelial cell (HUVEC) monolayers after 12 h of co-incubation under static (0 dyn/cm²) conditions (HUVECs + Bb, control). HUVECs+ (Bb + pFn): binding of bacteria pre-incubated with plasma fibronectin (pFn) to HUVECs. (HUVECs + pFn) + Bb: binding of bacteria to HUVEC pre-incubated with pFn. N = 3 independent bacterial and endothelial cultures/strain analyzed in 2 experiments. (b) Mean ± SEM bacterial interactions per minute with HUVEC under typical postcapillary venule shear stress condition (1 dyn/cm²). Videos of representative flow chamber experiments are presented in Supplementary Videos S1–S3. Numbers of bacteria that paused and moved more slowly over endothelia under flow were manually counted in time-lapses acquired at 15 frames per second (fps). Strains: Negative control strain: BBK32-deficient non-infectious adhesion-attenuated B31-A-derived strain (Parent; GCB706: Supplementary Table S1). Positive control strains: Parent expressing B. burgdorferi vascular adhesin BBK32 (Bb-BBK32); BBK32-expressing B31-derived infectious strain (Infectious; GCB726: Supplementary Table S1). Bb-Tp0751: Parent expressing Tp0751. N = 8 independent cultures per strain analyzed in 3 experiments. Statistics: one-way Kruskal-Wallis ANOVA with Dunn’s post-test (a), one-way ANOVA with Holm-Sidak post-test (b). *Indicates p < 0.05 vs. parent within incubation condition (a) and vs. parent (b).

Figure 2

Bond dissociation rates and interaction velocities of Tp0751-expressing B. burgdorferi. B. burgdorferi interactions with HUVEC in flow chambers at 0.5 and 1 dyn/cm² were captured by particle tracking of interaction trajectories (tracks) in time lapse sequences. (a) Sample time lapse projections depicting interaction tracks of individual bacteria (1 color/bacterium) captured at 1 dyn/cm² over 2 min. Scale bars: 38 µm. (b) The linear regression of the log-transformed number of interactions versus the lifetime (seconds) of the spirochete for 4 representative tracks (1 track for each of the 4 strains). The negative slope of each regression was an estimate of the dissociation rate (K_off) of the spirochete, as described in the Materials and Methods. (c) Mean ± SEM dissociation rates (K_off/off-rates) for individual load-bearing adhesion complexes mediating bacterial-endothelial interactions under flow. (d) Velocities of interacting bacteria (mean ± 95% CI). In (b–d), bond dissociation rates and interaction velocities were calculated from interactions obtained at both 0.5 and 1 dyn/cm². N = 4 and N = 8 independent flow chamber experiments for each strain at 0.5 and 1 dyn/cm², respectively. Eight replicate experiments were conducted at 1 dyn/cm² to obtain sufficient interaction numbers for tracking. Strains: Negative control strain: BBK32-deficient non-infectious adhesion-attenuated B31-A-derived strain (Parent; GCB706: Supplementary Table S1). Positive control strains: Parent expressing B. burgdorferi vascular adhesin BBK32 (Bb-BBK32); BBK32-expressing B31-derived infectious strain (Infectious; GCB726: Supplementary Table S1). Bb-Tp0751: Parent expressing Tp0751. Numbers of interactions analyzed/strain: Parent (408), Infectious (647), Bb-BBK32 (578), and Bb-Tp0751 (288). R² values for linear regressions in (b): >0.9. Statistics: one-way ANOVA with Holm-Sidak post-tests (c), one-way Kruskal-Wallis ANOVA with Dunn’s post-tests (d). *Indicates p < 0.05 vs parent. ⁺Indicates p < 0.05 vs Bb-BBK32.

Figure 3

Effect of Tp0751 on tethered and untethered interaction stability and velocity under shear stress. B. burgdorferi interacts with endothelia under PCV shear stress conditions by two mechanisms: untethered (dragging) interactions, and tethered interactions. Tethered interactions are stabilized by bungee cord-like tethers which anchor bacteria to endothelia, reduce force imposed on load-bearing adhesion complexes and increase adhesion bond stability (lifetime). (a,b) Proportion of total bacterial-endothelial interaction times during which bacterial length extended beyond the normal length, indicative of tether formation stabilizing the interaction ((a) % duration), and the average tether length (b) for all interactions at 0.5–1 dyn/cm². (c,d) Interaction complex dissociation rates for untethered (c) and tethered (d) interactions. Dotted lines indicate dissociation rates for non-specific interactions (non-adherent beads). (e,f) Velocities of untethered (e) and tethered (f) interactions. Summary values: mean ± 95% CI (a,b,e,f), mean ± SEM (c,d). Interaction properties were calculated from interactions obtained at both 0.5 and 1 dyn/cm². N = 4 and N = 8 independent flow chamber experiments for each strain at 0.5 and 1 dyn/cm², respectively. Strains: Negative control strain: BBK32-deficient non-infectious adhesion-attenuated B31-A-derived strain (Parent; GCB706: Supplementary Table S1). Positive control strains: Parent expressing B. burgdorferi vascular adhesin BBK32 (Bb-BBK32); BBK32-expressing B31-derived infectious strain (Infectious; GCB726: Supplementary Table S1). Bb-Tp0751: Parent expressing Tp0751. Numbers of interactions analyzed/strain (a,b): ≥288; (c,e): parent (42), infectious (127), Bb-BBK32 (130) and Bb-Tp0751 (39); (d,f): parent (366), infectious (520), Bb-BBK32 (448), and Bb-Tp0751 (249). Statistics: one-way Kruskal-Wallis ANOVA with Dunn’s post-tests (a,b,e,f); one-way ANOVA with Holm-Sidak post-tests (c,d). *Indicates p < 0.05 vs parent. ⁺Indicates p < 0.05 vs Bb-BBK32.

Figure 4

Bond strengths of Tp0751-dependent interactions under shear stress. Forces imposed on individual load-bearing adhesion bonds (F _b) for bacterial-endothelial interactions tracked at 0.5 and 1 dyn/cm² were estimated as described in the Materials and Methods. (a–c) Bond forces for all (a), untethered (b) and tethered (c) interactions (mean ± 95% CI). (d,e) Percentages of tracked interactions for each strain occurring at indicated F _b values. Percentages for both tethered and untethered interactions combined are shown in (d), and untethered interactions alone are shown in (e). F _b values for all interactions combined were binned by force range to obtain Mean ± SEM F _b values plotted on x-axes. For untethered interactions, interactions were grouped by F _b into a smaller number of bins than for all interactions combined (e), to provide sufficient interaction numbers in each bin for accurate calculation of bond dissociation rates at each bond force (f). For the parent strain and Bb-Tp0751, fewer than 25 interactions were obtained in all but the lowest force bin. For these strains, the data from the higher force bins were therefore pooled with values from the lowest force bin to obtain sufficient interaction numbers for dissociation rate calculations. This resulted in a single data point reflecting the values obtained for all untethered interactions for parent and Bb-Tp0751 strains. (f) Effect of increasing bond force on mean ± SEM bond dissociation rates. Dotted line indicates the dissociation rate for negative control beads. Strains: Negative control strain: BBK32-deficient non-infectious adhesion-attenuated B31-A-derived strain (Parent; GCB706: Supplementary Table S1). Positive control strains: Parent expressing B. burgdorferi vascular adhesin BBK32 (Bb-BBK32); BBK32-expressing B31-derived infectious strain (Infectious; GCB726: Supplementary Table S1). Bb-Tp0751: Parent expressing Tp0751. Numbers of interactions analyzed/strain: ≥288 (a,d); ≥39 (b,e,f); ≥249 (c). Statistics: one-way Kruskal-Wallis ANOVA with Dunn’s post-tests. *Indicates p < 0.05 vs parent. ⁺Indicates p < 0.05 vs BBK32.

Figure 5

Interaction of Tp0751-expressing B. burgdorferi with postcapillary venules in vivo. (a) Total bacterial interactions per minute with dermal PCVs of live mice, visualized by intravital microscopy (IVM). Representative IVM videos are presented in Supplementary Videos S4–S6. Numbers of bacteria that paused and moved more slowly over PCV surfaces under flow were manually counted in time-lapses acquired at 15 fps. (b) B. burgdorferi flaB DNA copy numbers in blood of mice collected after IVM, as measured by quantitative real-time PCR. (c) Copy number-adjusted interactions/min in PCVs. Numbers of mice (venules) analyzed per strain: parent: 14 (30); infectious: 8 (22); Bb-BBK32: 8 (24); Bb-Tp0751: 9 (25). Summary values represent means ± SEM for experiments performed with ≥4 independent bacterial cultures/strain. Statistics: one-way Kruskal-Wallis ANOVA with Dunn’s post-test. *Indicates p < 0.05 vs. parent.