Fig. 3: Antibodies against KpnO1 recognize the O1 antigen and differ in their capacity to drive complement activation.

a Antibody-dependent C3b deposition on KpnO1. KpnO1 pre-incubated with anti-KpnO1 IgG1 was incubated with 1% KpnO1∆NHS (NHS depleted from KpnO1 specific antibodies) as a complement source. C3b-deposition was detected using anti-hu-C3b-AF647 by flow cytometry. Potent complement activating antibodies in dark red (>2-fold increase in C3b deposition at 0.1 µg/ml, indicated by dotted line), moderate complement activating antibodies in light red and non-activating antibodies in gray. b Bars represent C3b deposition from (a) at 0.1 µg/ml IgG1, ordered based on C3b deposition intensity. c Antibody binding to KpnO1 was detected by flow cytometry and depicted with the same color coding as in (a). d Bars represent antibody binding from (c) at 0.1 µg/ml IgG1. e Antibody binding (at 1 µg/ml) to KpnO1 wild-type, O-antigen (∆wbbO) and D-galactan II (∆wbbY) was analyzed by flow cytometry. f Schematic representation of the LPS O1- and O2-antigen polysaccharide. The O1-antigen consists of D-galactan-II (orange) on top of D-galactan-I (blue), whereas the O2 antigen contains only D-galactan-I. Deletion of the WbbO and WbbY glycosyltransferase leads to the loss of the complete O-antigen and the loss of the distal D-galactan-II part, respectively¹⁶. g Antibody binding (at 1 µg/ml) to different O1-antigen expressing strains was analyzed using flow cytometry. The potent complement activating antibodies are highlighted by the red box (h) KpnO1 and an additional clinical strain expressing the O1-antigen (MB-17667) were lysed, and the proteins in the lysate were digested. Western blot was performed using different KpnO1 antibodies followed by goat anti-hu-IgG-HRP. Bands were visualized using ECL. a–e, g Flow cytometry data are represented by gMFI values of bacterial populations that were divided by the buffer control. g Data represent log2 transformed data. Data represent (a–d) mean ± SD or (e, g) mean of three independent experiments. h A representative experiment of two independent experiments is shown.