Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Abstract

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.

Figure 1

Specificity of mAb 2C7 and 2C7 reactivity against gonococcal strains used in this study. A. A simplified schematic of glycan organization of gonococcal LOS. Lipooligosaccharide glycosyl transferase (lgt) genes that are involved in LOS biosynthesis and are subject to phase variation are indicated in grey italic font. The grey shaded box represents the minimum glycan extensions expressed by naturally occurring gonococcal strains that are required for mAb 2C7 binding. B. Reactivity of mAb 2C7 with the LOS of gonococcal strains used in this study. Bacterial lysates were subjected to denaturing electrophoresis and Western blotting and membranes probed with tissue culture supernatants that contained mAb 2C7. Strain 24-1 was grown in media supplemented with CMP-NANA (2 µg/ml).

Figure 2

The alternative pathway of complement and properdin is required for killing of C4BP-binding strains of N. gonorrhoeae by mAb 2C7. A. Survival of gonococcal strains was measured in serum bactericidal assays in the presence of NHS alone, NHS plus mAb 2C7, or NHS plus mAb 2C7 where either factor B or properdin function was blocked with anti-factor Bb (α-Bb) or anti-properdin (α-P), respectively. Each data point represents the mean (±SD) of at least 2 independently repeated experiments. *, p<0.001 (2-tailed Student’s t-test) compared to the corresponding survival seen with mAb 2C7+NHS. B. C4BP binding to N. gonorrhoeae strains. N. gonorrhoeae strains 442089, 24-1 (LOS sialylated by growth in media containing CMP-NANA; labeled 24-1 sia+), FA1090, 15253, and 252 were incubated with 1%, 3% or 10% NHS and C4BP bound to the bacteria surface was measured by flow cytometry. One representative experiment of 3 independently performed experiments is shown. Numbers represent the average of the median fluorescence of each of the three experiments.

Figure 3

C3 deposition on, and factor B binding to N. gonorrhoeae. Bacterial strains were incubated with NHS that contained anti-C7 to prevent bacterial lysis, either in the presence or absence of mAb 2C7. Anti-factor Bb or anti-properdin mAbs to block function of these components was added to some reactions as indicated. C3 deposition on (upper graph) and factor B binding to (lower graph) bacteria was measured by whole cell ELISA. * indicates p≤0.001 compared to all other data points for the corresponding strain (2-tailed Student’s t test). C3 and factor B binding when bacteria were incubated with NHS alone were similar to results obtained when organisms were incubated with NHS + anti-C7 mAb (data not shown). Gonococcal H.8 antigen measured using mAb 2-8C-4-1 was similar across wells and ensured equal bacterial capture (data not shown). Controls where bacteria were incubated with heat-inactivated NHS showed OD_405nm readings <0.1 and have been omitted for simplicity.

Figure 4

Restoring C4BP binding to N. gonorrhoeae is associated with a properdin requirement for killing by mAb 2C7. The PorB.1A molecule of strain 252 (binds C4BP minimally) was replaced with the PorB.1A from strain FA19 (strong C4BP binder; Ref. (43)) to yield mutant strain 252/PorFA19. A. Binding of C4BP to 252/PorFA19. Bacteria were incubated with increasing concentrations of NHS as indicated and bound C4BP was measured by flow cytometry. Deficient C4BP binding to parent strain 252 incubated with 10% NHS is shown as the Control. Numbers adjacent to each histogram represent the average of the median fluorescence of at least two independently performed experiments. B. 2C7 epitope expression by the LOS of mutant strain 252/PorFA19. Western blotting was performed as described in Fig 1A. Parent strain 252 was used as a control. C. Functional properdin is required for 2C7-dependent killing of C4BP-binding isogenic mutant strain 252/PorFA19. Bacteria were incubated with NHS alone, 2C7 + NHS or 2C7 + NHS + α-P (anti-properdin mAb). Bacterial survival following incubation of the reaction mixture for 30 min at 37 °C was measured in a serum bactericidal assay. Controls included parent strain 252 (binds C4BP minimally) and strain FA1090 (strong C4BP binder). D. C3 deposition on 252/PorFA19. Bacteria were incubated with NHS containing anti-C7, either in the presence or absence of mAb 2C7. Where indicated, properdin function was blocked with an anti-properdin mAb. *, p<0.001 (2-tailed Student’s t test).

Figure 5

Properdin is required for killing of C4BP-binding strains of N. gonorrhoeae by immune human antibody directed against the 2C7 LOS epitope. A. Bactericidal activity of gonococcal vaccine-elicited human immune serum (directed against the 2C7 LOS epitope) requires expression of lactose on HepII (Fig. 1A). Strain 15253 and its isogenic lgtG deletion mutant (15253 ΔlgtG) were incubated with NHS alone (indicated as 0 on the graph) as a constant source of complement and with increasing concentrations of heat-inactivated vaccine-elicited human immune serum (complement inactivated, but antibodies intact); bacterial survival following a 30 min incubation period was measured. B. Properdin-dependent killing of C4BP-binding N. gonorrhoeae by vaccine-elicited human immune serum. C4BP-binding strains FA1090 and 15253, and minimal C4BP-binding strains 252 and sialylated 24-1 (24-1 sia+), were incubated with NHS alone, NHS + immune human serum (final concentration 6.7%) or NHS + immune human serum + α -P (anti-properdin mAb). Bacterial survival was measured following incubation of reaction mixtures for 30 min at 37 °C. Each data point represents the mean (±SD) of 2 separate experiments.

Figure 6

Killing of C4BP-binding gonococcal strain FA1090 by anti-OMV antibody requires functional properdin. A. Broad cross-reactivity of murine antiserum raised against a FA1090 membrane preparation (“Memb. prep.”). The membrane preparation that was used as the immunogen (a silver stain of the same is also shown) and whole cell lysates of FA1090 and 252 were separated on a 4–12% Bis-Tris gel, transferred to a PVDF membrane that was then overlaid with a 1:1000 dilution of anti-FA1090 OMV in PBS/0.05% Tween 20. Mouse IgG bound to bacterial antigens was disclosed with anti-mouse IgG alkaline phosphatase and substrate. Localization of IgG- reactive PorB.1B (faintly visualized only in the membrane preparation and FA1090 lanes) and Opacity protein (Opa) bands are indicated. LOS and LOS-reactive IgG bands are indicated with a solid black dots. B. Murine anti-OMV serum antibody (dilutions ranging from 1/75 to 1/300) mediates complement-dependent killing of strains FA1090 (left panel) and 252 (right panel) in the presence (dashed lines) or absence (solid lines) of α-P (anti-properdin mAb) that blocks function of properdin; 16.7% NHS was used as a source of human complement. The 1/75 dilution experiment represents the mean (±SD) of 3 separately performed experiments; the 1/150 and 1/300 dilution experiments, the mean (±SD) of 2 experiments. *, p<0.005 (2-tailed Student’s t-test).