Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H

Abstract

Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.

FIGURE 1.

A, binding of human and chimpanzee fH domains 18–20/Fc fusion proteins to sialylated N. gonorrhoeae. Binding of HufH18–20/Fc is indicated by the solid line and (lack of) binding of ChfH18–20/Fc by the dashed line. Lack of binding of protein HufH18–19/ChfH20/Fc (HufH20 replaced by ChfH20) is shown by the shaded gray histogram. In all graphs, the x axis represents fluorescence on a log₁₀ scale, and the y axis represents the number of events. The control graph (no fH/Fc protein added) is depicted by the faint dotted line. One representative experiment of three independently performed experiments is shown. B, inhibition of binding of full-length HufH to sialylated N. gonorrhoeae by HufH18–20/Fc and ChfH18–20/Fc. Sialylated N. gonorrhoeae strains 252 (PorB.1A) and F62 (PorB.1B) were incubated with 0.5 μg of full-length HufH and either ≈ 1.5, 3, or 6× molar excess (compared with full-length HufH) of purified HufH18–20/Fc (left panel) or ChfH18–20/Fc (right panel). Full-length HufH that bound to bacteria was detected with mAb 90× (28), followed by anti-mouse IgG (Fab-specific) FITC. In all graphs, the x axis represents fluorescence on a log₁₀ scale, and the y axis represents the number of events. No HufH or fusion protein was contained in the reaction mixtures represented by the “Control” histograms. One representative experiment is shown of two independently performed experiments.

FIGURE 2.

Binding of HufH18–20/Fc to sialylated N. gonorrhoeae: effect of human-to-chimpanzee mutations in HufH domain 20. A, amino acid sequence comparison of human and chimpanzee fH domains 20 (fH20). Identical amino acid residues are denoted by dots. B, binding to sialylated gonococcal strains 252 and F62 by HufH18–20/Fc constructs that separately contained the indicated chimpanzee fH domain 20 point mutations relative to HufH18–20/Fc wild-type. The bar graph labeled Control contains no fusion proteins. In all bar graphs, the x axis represents each mutant, and the y axis represents the percentage of binding relative to WT using median fluorescence. Values represent the mean calculated from two or more independently performed experiments ± S.E.

FIGURE 3.

Binding of HufH18–19/ChfH20/Fc (HufH20 replaced by ChfH20) constructs to sialylated N. gonorrhoeae. Amino acid residues at positions 1203 and 1217 in ChfH20 were individually substituted by their human counterparts. In all graphs, the x axis represents fluorescence on a log₁₀ scale, and the y axis represents the number of events. The graph labeled HufH18–20/Fc serves as the positive control, and HufH18–19/ChfH20/Fc and Control, the latter containing no fusion proteins, serve as negative controls. One representative experiment of two independently performed experiments is shown.

FIGURE 4.

Heparin inhibits binding of HufH18–20/Fc to sialylated gonococci. Binding of HufH18–20/Fc to sialylated 252 (PorB.1A) and F62 (PorB.1B) strains was measured by flow cytometry in the presence of heparin (final concentrations of 100 or 400 units (u)/ml; denoted by the dashed line and shaded gray histograms, respectively). Binding of HufH18–20/Fc in the absence of heparin is indicated by the solid line and Control (no HufH18–20/Fc added) by the dotted line. The x axis represents fluorescence on a log₁₀ scale, and the y axis represents the number of events. One representative experiment of three independently performed experiments is shown.

FIGURE 5.

Comparison of the crystal structure of HufH domains 19–20 (31) (A) with a molecular model of fH domains 19–20 with the R1203N mutation (B). In both A and B, surface representation of charge is shown with colors; red indicates electronegative, and blue indicates electropositive regions (range from −3 to +3 kT/e). The site of the R1203N mutation is circled. The model (B) indicates that the effect of the R1203N mutation upon surface charge is limited to the immediate area surrounding the mutated residue, i.e. the encircled area only. Location of Thr-1217 is indicated in the model structure with a black arrow.

FIGURE 6.

Inhibition of binding of HufH to sialylated gonococci results in reversal of inhibition (killing) of N. gonorrhoeae by the alternative pathway of complement in NHS. Killing of sialylated gonococcal strains F62 (PorB.1B; left panel) and 252 (PorB.1A; right panel) in 10% NHS containing increasing amounts of HufH18–20/Fc or HufH18–20/Fc mutant proteins: HufH18–20/Fc (filled diamonds), HufH18–19/ChfH20 N1203R/Fc (open squares) and HufH18–20 R1203A/Fc (filled triangles). The x axis represents the concentration of purified recombinant HufH18–20/Fc proteins added to reaction mixtures that contained 10% NHS; the y axis indicates the percent (%) killing. The numbers in the box represent the molar ratio of recombinant fH domains 18–20/Fc to endogenous fH in NHS (10%) at different concentrations of the recombinant domains used. Results represent the mean killing calculated from three or more independently performed experiments ± S.E.