Fig. 2: Antibodies targeting the O2-antigen but not the capsule drive complement activation on KpnO2. | Nature Communications

Fig. 2: Antibodies targeting the O2-antigen but not the capsule drive complement activation on KpnO2.

From: Agnostic B cell selection approach identifies antibodies against K. pneumoniae that synergistically drive complement activation

Fig. 2

a Antibody-dependent C3b deposition on KpnO2. Bacteria were pre-incubated with a concentration range of KpnO2 IgG1s, followed by addition of 2% KpnO2∆NHS (NHS depleted from KpnO2 specific antibodies) as a complement source. C3b-deposition was detected using anti-hu-C3b-AF647 by flow cytometry. Potent complement activating antibodies in dark blue (more than 2-fold increase of C3b signal at 0.1 µg/ml indicated by the dotted line), moderate activating antibodies in light blue antibodies and non-complement activating antibodies in gray. b Bars represent C3b deposition from (a) in the condition where 0.1 µg/ml IgG1 was added, ranked on C3b deposition level. c Antibody binding of the different KpnO2 specific antibodies to KpnO2 was detected by flow cytometry and depicted with same color coding as in (a). d Bars represent antibody binding data from (c) at 0.1 µg/ml IgG1. e, f KpnO2 transposon mutants that are not recognized by (e) UKpn1 or (f) UKpn2 were single cell sorted. The barcode of the selected mutants was sequenced to determine the location of the transposon insertions (indicated as black bars). e Mutants that were no longer bound by UKpn1 had transposon insertions in genes involved in capsule synthesis. f Mutants that were not recognized by UKpn2 transposon had insertions in the rfb locus. g Antibody binding (at 1 µg/ml) to capsule (∆wbaP) and O-antigen (∆wbbO) deletion mutants was measured by flow cytometry and depicted with the same color coding as used in (a), larger dots indicate UKpn1 (gray) and UKpn2 (dark blue). ad, g Flow cytometry data are represented as relative gMFI of bacterial populations by dividing values by the isotype control. ad Data represent mean ± SD of three independent experiments and (g) average of 2-3 independent experiments.

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