Figure 1

IgM from serum can trigger classical pathway-mediated killing of E. coli MG1655. (a) Schematic representation of the purified classical pathway assay using the membrane impermeable DNA dye Sytox (blue stars) as readout for inner membrane damage. In the first step, bacteria are mixed with C1-complex, and antibodies for 15 min at 4 °C. In the second step that follows without washing, C1-inhibitor and C2–9 are added for 45 min at 37 °C. (b) Bacterial inner membrane damage (percentage Sytox positive) of E. coli MG1655 bacteria that were incubated with a concentration range of complement components, with either no antibody added (No Ab), 100 µg/mL polyclonal IgM, or 100 µg/mL polyclonal IgG. The concentrations of complement components correspond to the indicated serum percentages. As a positive control, bacteria were incubated with a concentration range NHS for 45 min at 37 °C. (c) Bacterial viability (CFU/mL) of MG1655 after incubation with the purified CP assay with either no antibody added (No Ab) or 100 µg/mL polyclonal IgM. At timepoint 0 min, a sample was taken as is shown as a dotted line with T = 0. The detection limit of the assay is also shown as a dotted line. (d) Percentage of the total MG1655 bacterial population that became positive for the DNA dye Sytox blue as measured with flow cytometry. Bacteria were treated either with RPMI, 100 µg/mL polyclonal IgM, 1.25% serum equivalent complement components without antibody, 1.25% serum equivalent with 100 µg/mL IgM (all present), or 1.25% serum equivalent with 100 µg/mL polyclonal IgM without one single indicated complement component. Data represent mean ± SD of three independent experiments (b, c, and d).