Figure 5.

Membrane attack complex (C5b-8/C5b-9) on the surface of clinical A. baumannii isolates. 10⁶ CFU of bacteria was incubated with either 25% normal human sera or 25% heat-inactivated human sera, followed by incubation with the mouse monoclonal antibody aE11 (Abcam) that recognizes a neo-epitope on polymeric C9, and detected using an anti-mouse IgG conjugated to allophycocyanin (APC) (Jackson Immunoresearch). (A) Bar graph showing the C5b-8/C5b-9 deposition index (% positive * Median fluorescent intensity of bacteria staining positive for C5b-8/C5b-9 deposition). (B) Representative histograms showing the deposition of C5b-8/C5b-9 on 1x10⁶ CFU of three A. baumannii isolates. Each graph shows a grey histogram that represents bacteria labelled with primary and secondary antibody only, black-dotted line represents bacteria incubated with heat-inactivated human sera and black solid line represent bacteria incubated with normal human sera. (C) Confocal imaging showing C5b-8/C5b-9 deposition on a subset of A.baumannii clinical isolates following incubation with heat-inactivated sera (top panel) or normal sera (bottom panel). Blue represent DAPI stained bacteria and green shows bacteria positive for MAC deposition. Figures below each panel represent the percentage of bacterial cells in each panel showing atleast some green fluorescent staining representing MAC deposition. A scale bar equivalent to 10 µm is indicated by a white line in all images. (D) Correlation between C5b-8/C5b-9 deposition index and C3b/iC3b deposition on the nine clinical A. baumannii isolates. Bars represent mean values for each strain and the error bars indicate standard deviations (SDs) (n = 5). T-test was used for statistical analysis *: p-value < 0.05, ***: p-value < 0.001, ns: p-value > 0.05. Pooled data from two independent experiments is shown.