Figure 8
Reactivity of IsdB antibodies from human sera and their effect on IsdB-mediated bacterial adhesion to HUVEC monolayers. (A) Patients’ sera reactivity for IsdB protein. Microtiter wells coated with IsdB NEAT1-NEAT2 were probed with IgG isolated from sera of patients with staphylococcal endocarditis. IgG from healthy donors were used as controls. Bound antibody was detected by the addition of rabbit anti-human HRP-conjugated IgG to the wells. Data are expressed as means ± S.D. of triplicate tests. (B) Effect of IgG isolated from patients’ sera on IsdB NEAT1-NEAT2 binding to HUVEC monolayers. Confluent HUVEC monolayers were treated with calcium ionophore A23187 and incubated with recombinant IsdB NEAT1–NEAT2 in the presence of the indicated IgG isolated from patients’ sera. Bound IsdB NEAT1–NEAT2 was detected by the addition of a rabbit polyclonal IsdB antibody followed by HRP-conjugated goat anti-rabbit IgG. The binding of IsdB NEAT1–NEAT2 to the monolayers in the presence of control IgG is also reported. Binding observed in the absence of antibodies was set as 100% binding. (C) Adhesion of S. aureus SH1000 to HUVEC monolayers in the presence of IgG isolated from patients’ sera. HUVEC cell monolayers treated with ionophore A23187 were incubated with cells of S. aureus SH1000 WT in the presence of the indicated IgG isolated from patients’ sera or healthy human sera. Adhesion was determined by the addition of an HRP-conjugated rabbit anti-mouse IgG. Bacterial attachment observed in the absence of antibodies was set as 100% adhesion. Statistically significant differences are indicated (*, P < 0.05; **, P < 0.01). (D) Adhesion of L. lactis ectopically expressing IsdB to HUVEC monolayers in the presence of IgG isolated from patients’ sera. HUVEC cell monolayers treated with ionophore A23187 were incubated with cells of L. lactispNZ8037 or L. lactispNZ8037::isdB in the presence of the indicated IgG isolated from patients’ sera or healthy human donors. Adhesion of bacteria to HUVEC monolayers was detected through rabbit anti-L. lactis IgG followed by an HRP-conjugated goat anti-rabbit IgG. Statistically significant difference is indicated (***, P < 0.001). Bars reported in (B–D) represent means ± SD of triplicate tests.
