FIG 4.

Tp0751-specific serum disrupts T. pallidum interactions with endothelial cells. (A) Representative immunofluorescent images from the T. pallidum serum inhibition adhesion assay showing HUVEC nuclei (blue; DAPI), cellular margins (green; anti-VE-cadherin), and T. pallidum (red; anti-FlaA). Quantification of HUVECs and T. pallidum were compared between anti-Tp0751 preincubations (top row) and anti-Tp0327 preincubations (bottom row). Zoomed images reveal comparisons of treponemal abundance (red; anti-FlaA) between anti-Tp0751 and anti-Tp0327 treatments. (B and C) Number of adherent T. pallidum (Tp) per HUVEC following preincubation with anti-Tp0327 serum (blue, negative control) or Tp0751-specific serum (red). In panel B, the mean number of T. pallidum per HUVEC plus SEM was quantified using immunofluorescence in a blind manner from five fields of view (FOV) in duplicate wells in four independent experiments. Statistical analysis was performed by Student’s two-tailed t test comparing endothelial binding of T. pallidum preincubated with Tp0751-specific serum to T. pallidum preincubated with Tp0327-specific serum (P = 0.025). In panel C, quantitative real-time PCR (qPCR) measured flaA DNA concentration from T. pallidum-endothelial adhesion assays. Results were normalized to HUVEC gDNA concentration by quantifying GAPDH and presented as mean flaA copy number per picogram of GAPDH DNA from duplicate wells in three independent experiments. Statistical analysis was performed by unpaired two-tailed t test with Welch’s correction comparing endothelial binding of T. pallidum preincubated with Tp0751-specific serum to T. pallidum preincubated with Tp0327-specific serum (P = 0.023).