Fig 5. Tp0751 peptide 10 inhibits adhesion of Tp0751-expressing B. burgdorferi to HUVEC monolayers.

(A) Schematic showing the experimental design of the competitive inhibition assay. Confluent HUVEC monolayers seeded on artificial basement membrane were preincubated with synthetic Tp0751 peptides (p4, p6, p10, p11, p4scr, p6scr, and p10scr1) to allow for peptide-endothelial cell interactions. HUVECs were then co-incubated with parent (negative control) or Tp0751-expressing B. burgdorferi strains to assess the competitive inhibition capacity of the synthetic Tp0751 peptides. After washing to remove non-adherent bacteria, B. burgdorferi adhesion to HUVECs was quantified using fluorescence microscopy to count the number of adherent bacteria per field of view (FOV) at 400x magnification. (B) Bar graph illustrating the number of adherent B. burgdorferi per FOV. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. For statistical analyses, attachment by strain Bb-Tp0751 to HUVEC monolayers preincubated with peptides p4, p6, and p10 was compared to the attachment of strain Bb-Tp0751 in the presence of corresponding scrambled peptides, using the Student’s two-tailed t-test. Strain Bb-Tp0751 exhibited statistically significant lower levels of binding to HUVEC monolayers preincubated with peptide p10 (*p≤0.005) when compared to the levels of binding when preincubated with the scrambled version of peptide 10 (p10scr1). Bb-Tp0751 adhered significantly more to endothelia than the parent under untreated conditions and when monolayers were pre-incubated with all other peptides (p˂0.05). The parent and Bb-Tp0751 strains are indicated by clear white bars and striped colored bars, respectively. (C) Line graph representing the effect of increasing concentrations (0 nM, 0.54 nM, 5.45 nM, 54.5 nM, and 545 nM) of p10 and negative control peptide p8 on adherence of Parent and Bb-Tp0751 to HUVECs. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. Statistical analysis, was performed using the Student’s two-tailed t-test. Strain Bb-Tp0751 exhibited significantly lower levels of binding to HUVEC monolayers when preincubated with ≥ 5.45 nM p10 compared to the levels of binding when preincubated with ≥ 5.45 nM p8 (*p≤0.005). A non-linear regression curve was fitted and the IC50 for p10 inhibition of HUVEC binding by Bb-Tp0751 was calculated (IC50 = 17 nM) using GraphPad Prism data analysis software (San Diego, CA).