A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae

Abstract

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.

Figure 1

Schematic representation of rH and the nine deletion mutant molecules used in this study. Each circle represents an individual SCR. rH and rH constructs lacking the indicated domains were generated by PCR, expressed in insect cells using the baculovirus system, and purified for use in this study (7, 49).

Figure 2

Flow cytometric quantitation of fH binding (using affinity-purified polyclonal rabbit anti–human fH) to sialylated strain 24-1 NANA (top) and parent strain 24-1 (bottom) when opsonized for 30 min with pooled NHS (10 μl), HIS (10 μl), or purified human fH (5 μg). 24-1 NANA showed significantly greater fH binding under all three conditions of opsonization.

Figure 3

Western blot confirming increased fH binding to 24-1 NANA compared to 24-1 (detected by sheep anti–human fH), when incubated with NHS, HIS, or purified fH.

Figure 4

Quantitation of C3b, iC3b, factor Bb, and total C3 binding to 24-1 and 24-1 NANA after incubation in NHS for 10 min, determined by whole cell ELISA. Each bar represents the mean ± SD of two experiments. The ratio of C3b to iC3b binding was significantly greater for 24-1 than 24-1 NANA, which bound almost no C3b. 24-1 NANA bound ∼10-fold less Bb and 4-fold less total C3 than 24-1 (inset).

Figure 5

Binding of rH and the nine rH mutant molecules to 24-1 NANA in a flow cytometry assay. All molecules that contained SCRs 16–20 bound to the organism. Only rHΔ16-20 and rHΔ11-20 did not show significant binding, suggesting that SCRs 16–20 are essential for fH binding to 24-1 NANA.

Figure 6

Specificity of fH binding to sialic acid is shown by neuraminidase treatment of 24-1 NANA both before (A, Neu→ fH) and after (B, fH→ Neu) incubating the organisms with pure fH. In both experiments, fH binding is reduced to near background levels. The solid lines represent fH binding to 24-1 NANA (positive control), the shaded areas represent fH binding to neuraminidase-treated samples, and the dotted lines show isotype controls. No binding of fH above isotype control levels was seen with 24-1 (data not shown).

Figure 6

Specificity of fH binding to sialic acid is shown by neuraminidase treatment of 24-1 NANA both before (A, Neu→ fH) and after (B, fH→ Neu) incubating the organisms with pure fH. In both experiments, fH binding is reduced to near background levels. The solid lines represent fH binding to 24-1 NANA (positive control), the shaded areas represent fH binding to neuraminidase-treated samples, and the dotted lines show isotype controls. No binding of fH above isotype control levels was seen with 24-1 (data not shown).

Figure 7

Specificity of neuraminidase treatment of gonococcal LOS. Samples used in flow cytometry (Fig. 6) were centrifuged, and then the pellets were harvested, digested, and subjected to electrophoresis on a 15% SDS-polyacrylamide gel followed by Western blotting. Blots were probed with mAb 3F11 that specifically recognizes the sialylation epitope (Galβ1→ 4GlcNAc) on LOS. Binding of 3F11 is abolished when LOS is sialylated, and is restored with neuraminidase treatment. Lane 1: 24-1 NANA incubated with fH alone; lane 2: empty; lane 3: 24-1 NANA treated with neuraminidase before incubation with fH (Neu→ fH); lane 4: 24-1 NANA treated with neuraminidase after incubation with fH (fH→ Neu).