Journal: Microbiology Spectrum

Article Title: Transendothelial migration of the Lyme disease spirochete involves spirochete internalization as an intermediate step through a transcellular pathway that involves Cdc42 and Rac1

doi: 10.1128/spectrum.02221-24

Figure Lengend Snippet: Transendothelial migration of B. burgdorferi in Transwell chambers. (A) To study B. burgdorferi transmigration, Transwell chambers were seeded with hMVEC-d or hTERT cells until a tight monolayer was formed (<4% albumin diffusion). The upper chamber was infected with 3 × 10 5 spirochetes; after 20 h of infection, we counted the spirochetes in both the upper and lower chambers (either manually or by flow cytometry) and calculated the percentage of total transmigrated spirochetes (lower chamber). ( B, C ) The graphs show the percentage (mean ± SD) of B. burgdorferi that had transmigrated through human microvascular endothelial cells as determined by counting spirochetes in both the upper and lower chamber by flow cytometry. The data represent the mean of % transmigration ±SD of three independent experiments performed in quadruplicate and analyzed for significance using the Mann–Whitney test. NI denotes the non-infectious strain GCB705 (B31-A + pTM61 gent,gfp, see ), and WT indicates GCB726. ( D ) Evaluation of B. burgdorferi transendothelial migration in hTERT treated with AKB-9785. The complete chamber was treated with 5 µM AKB9785 for the duration of the assay. The upper chamber was infected with 3 × 10 5 spirochetes, and the percentage of total transmigrated spirochete was assessed by counting in a Petroff–Hausser chamber and dark-field microscopy after 20 h of infection. The data represent the mean % transmigration ±SD of three independent experiments performed in quadruplicate; non-parametric ANOVA was performed to compare the control cells with the treated cells using the Kruskal–Wallis post-test (ns = not significant). ( E ) Demonstration of the effectiveness of AKB-9785 to lock intercellular junctions in hTERT cells. Ly 294002 at 40 µM was used to disrupt the monolayer, and AKB-9785 was used to lock intercellular junctions and preserve monolayer integrity, which was assessed using an albumin diffusion assay: 10 μg of 555-Alb was added to the upper chamber at 16 h, and the reading was carried out at the final point (20 h). The graph represents the mean ± SD of three experiments, with 1–4 samples for each experimental condition. Statistics were evaluated with the Kruskal–Wallis test and Dunn’s multiple comparison; P < 0.05 was considered significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: Primary human dermal microvascular endothelial cells (hMVEC-d) were purchased from Lonza (CC-2543), grown in Basal Medium (EBM-2) (CC-3156, Lonza) complete media (with supplements EGMTM-2 SingleQuotsTM Supplements [CC-4176, Lonza]) at 37°C under 5% CO 2 and used before passage five. hTERT-immortalized dermal microvascular endothelial cell, neonatal (CRL4060, ATCC), was used. hTERT was cultured in Vascular Cell Basal Medium (VCBM) (PCS-100-030, ATCC) with the microvascular endothelial cell growth kit-BBE (PCS-110-040, ATCC) +0.5 μg/mL puromycin (P8833, Sigma-Aldrich) according to the manufacturer’s instruction.

Techniques: Migration, Transmigration Assay, Diffusion-based Assay, Infection, Flow Cytometry, MANN-WHITNEY, Microscopy, Control, Comparison