Treponema pallidum protein Tp0136 promotes angiogenesis to facilitate the dissemination of Treponema pallidum

Abstract

The incompletely eliminated Treponema pallidum (T. pallidum) during primary syphilis chancre infection can result in the progression of secondary, tertiary, or latent syphilis in individuals, suggesting that T. pallidum has successfully evaded the immune response and spread to distant sites. The mechanism underlying the dissemination of T. pallidum is unclear. Here, a syphilitic rabbit model dorsal-injected with recombinant Tp0136 protein or Tp0136 antibody subcutaneously was used to demonstrate the role of Tp0136 protein in promoting the dissemination of T. pallidum to the testis and angiogenesis in vivo; vascular endothelial cell line HMEC-1 was employed to display that Tp0136 protein enhances the angiogenesis. Furthermore, the three-dimensional microfluidic angiogenesis system showed that the angiogenesis would heighten vascular permeability. Then transcriptome sequencing analysis, in conjunction with cell-level validation, elucidated the critical role of the PI3K-AKT signaling pathway in the promotion of angiogenesis by Tp0136 protein, resulting in heightened permeability. These findings elucidate the strategy employed by T. pallidum in evading immune clearance.

Keywords: PI3K-AKT; Treponema pallidum; Treponema pallidum protein Tp0136; angiogenesis; vascular permeability.

Figure 1.

Tp0136 accelerated dissemination of T. pallidum. (a) Procedure of the rabbit infection experiment. The number of repetitions for each processing method can be found in the figure indicated by the numeric value of “n”. (b) Dynamic changes in the T. pallidum load in skin lesions on days 18, 33, 36, 42, and 60 in different groups. At each time point, three rabbits are used as biological repetitions for T. pallidum load detection. (c) Dynamic changes in the T. pallidum load in testes on days 18, 33, 36, 42, and 60 in different groups. At each time point, three rabbits are used as biological repetitions for T. pallidum load detection. (d) Dynamic changes in the T. pallidum load in skin lesions on days 42, 51, and 60 in the different anti-Tp0136 subgroups. At each time point, three rabbits are used as biological repetitions for T. pallidum load detection. (e) Dynamic changes in the T. pallidum load in testes on days 42, 51, and 60 in the different anti-Tp0136 subgroups. At each time point, three rabbits are used as biological repetitions for T. pallidum load detection. Data are expressed as the mean ± standard deviation (SD) from three independent experiments. Two-way analysis of variance (ANOVA) was used to compare the mean values of three or more groups with two independent variables. ***P < 0.001, ns; nonsignificant difference.

Figure 2.

Tp0136 promoted angiogenesis in vivo and in vitro. (a) Effect of Tp0136 protein and antibody on blood vessels in lesions. Red arrows indicate the blood vessels. The bar graph indicates the number of the blood vessels. Scale bars = 100 mm. (b) Effect of Tp0136 on cell migration using transwell migration assay. The bar graph indicates the crystal violet area. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (c) Effect of Tp0136 on spheroid-based sprouting. The bar graph indicates the number of sprouting. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (d) Effect of Tp0136 on tubule formation. Tp0136 (10 mg/mL) was coincubated with HMEC-1 cells for 6 h, after which tubule formation was stained using Calcein AM. Bar graphs indicate the number of nodes, the number of meshes, and the total branching length. Scale bars = 500 mm. The experiment was repeated three times independently for each group. (e-f) Three-dimensional microfluidic angiogenesis system of angiogenesis processed by Tp0136. (g) Effect of Tp0136 on three-dimensional angiogenesis. The bar graph indicates the number and the average length of angiogenic sprouts. Phalloidin-iFluor 555-red, DAPI-blue. Scale bars = 100 mm. The experiment was repeated three times independently for each group. Data are expressed as the mean ± SD from three independent experiments. Student's t test was used to compare data between two groups. One-way ANOVA was used to compare the mean values of three or more groups with one independent variable. *P vs. Control, *, P<0.05, **, P<0.01, ***, P<0.001.

Figure 3.

Tp0136 increased vascular permeability with angiogenesis. (a) Effect of Tp0136 on vascular permeability detected using Evans blue-bovine serum albumin. Left: Vascular permeability in HMEC-1 cells stimulated with different concentrations of Tp0136 for 6 h. Right: Vascular permeability in HMEC-1 cells incubated with 10 mg/mL Tp0136 for different durations. The experiment was repeated three times independently for each group. (b) Effect of Tp0136 on vascular permeability detected using three-dimensional angiogenesis analysis. The pseudo-colored fluorescent images after 0 and 8 min after the addition of the dextran solutions. Time is indicated in h, min, and s. Data are expressed as the mean ± SD from three independent experiments. One-way ANOVA was used to compare mean values of three or more groups with one independent variable. * P < 0.05, ** P < 0.01, *** P < 0.001, ns; nonsignificant difference.

Figure 4.

Expression of PI3K/AKT signaling pathway in Tp0136-induced angiogenesis. (a) GO analysis of the first 3000 differentially expressed genes after Tp0136 treatment in HMEC-1 cells. (b) KEGG pathway analysis of the first 3000 differentially expressed genes after Tp0136 treatment in HMEC-1 cells. (c) Expression of the PI3K, p-PI3K, AKT, and p-AKT proteins in HMEC-1 cells stimulated with different concentrations of Tp0136 revealed using western blotting. The bar graphs on the right indicate the ratios of the phosphorylated proteins versus the total proteins. The experiment was repeated three times independently for each group. (d) Expression of the PI3K, p-PI3K, AKT, and p-AKT proteins in HMEC-1 cells stimulated with Tp0136 for different durations using western blotting. The experiment was repeated three times independently for each group. Data are expressed as the mean ± SD from three independent experiments. Two-way ANOVA was used to compare the mean values of three or more groups with two independent variables. *P vs. Control, * P < 0.05, ** P < 0.01, *** P < 0.001, ns; nonsignificant difference.

Figure 5.

Tp0136 promoted angiogenesis via PI3K/Akt leading to the increase in vascular permeability. (a) Effect of LY294002 on Tp0136-induced tubule formation. Scale bars = 500 mm. Bar graphs indicate the number of nodes, the number of meshes, and the total branching length in tubule formation. The experiment was repeated three times independently for each group. (b) Effect of LY294002 on Tp0136-induced angiogenesis was analyzed using three-dimensional angiogenesis analysis. The bar graph shows the number and the average length of angiogenic sprouts. Phalloidin-iFluor 555-red, DAPI-blue. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (c) Effect of LY294002 on Tp0136-induced vascular permeability in three-dimensional microfluidic angiogenesis system. Time is indicated in h, min, and s. Data are expressed as the mean ± SD from three independent experiments. One-way ANOVA was used to compare the mean values of three or more groups with one independent variable. * P vs. Control, ** P < 0.01, *** P < 0.001. # P vs. Tp0136, # P < 0.05, ## P < 0.01.

Figure 6.

Treponema pallidum protein Tp0136 actively promotes the dissemination of T. pallidum by increasing vascular permeability. Tp0136 accelerated the dissemination of T. pallidum, and Tp0136 facilitated angiogenesis via PI3K-AKT leading to increased vascular permeability favoring the dissemination of T. pallidum.