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. 2009 Apr 17;191(12):3861–3868. doi: 10.1128/JB.01313-08

FIG. 5.

FIG. 5.

N. meningitidis bacteria attached to host cells are protected from LL-37. (A) Schematic representing early-, mid-, and late-stage treatments with LL-37 for the adhesion assay. (B) FaDu cells were infected with strain FAM20 (multiplicity of infection = 5) and treated with LL-37 (at 0 and 10 μM). control, no LL-37 treatment; early, epithelial cells were treated with LL-37 for 30 min and then thoroughly washed before addition of bacteria for 2 h; mid, bacteria and LL-37 were added to epithelial cells at the same time for 2 h; late, bacteria were added to epithelial cells to attach for 1 h and thoroughly washed before LL-37 was added for an additional 2 h. Cells were thoroughly washed and viable counts of adherent bacteria determined. Data represent mean percentages of recovery (treated) compared to control results (untreated) ± standard errors (n = 3). (C) FaDu cells were incubated with LL-37 (at 0 and 10 μM) for 30 min at 4°C, washed, stained with a biotinylated anti-LL-37 antibody, and analyzed by flow cytometry. Representative histogram shows LL-37 binding to FaDu cells after incubation with LL-37. (D) FAM20 and LL-37 (at 0 and 10 μM) were added to plastic microtiter plates. control, no LL-37 treatment; mid, bacteria and LL-37 were added to plates at the same time for 2 h; late, bacteria were added to plates to attach for 1 h and thoroughly washed before LL-37 was added for an additional 2 h. Plates were thoroughly washed to remove unbound bacteria and viable counts of adherent bacteria determined. Data represent mean percentages of recovery (treated) compared to control results (untreated) ± standard errors (n = 3). (E) Strain FAM20 and PilT mutant bacteria, after being resuspended to form 105 CFU/ml, were incubated with 0 to 16 μM LL-37, and viable counts were determined after 3 h. Data represent mean survival ratios ± standard errors (n = 3). (F) FaDu cells were infected with FAM20ΔPilT as described for panel B. * and ** represent statistically significant differences (P ≤ 0.01 and P ≤ 0.05, respectively).