Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae

Abstract

We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

Figure 1

Demonstration of cofactor activity on gonococcal strains that bind C4bp. Strains F62, MS11, and FA19 were incubated with 20% NHS for 30 min at 37°C, followed by detection of intact C4b fragments remaining on the bacterial surface by flow cytometry. C4bp cofactor activity would result in factor I–mediated cleavage of C4b to C4c and C4d. C4d remains bound to the bacterial surface, whereas the C4c fragment is released into solution. Anti-C4d mAb recognizes both C4b as well as C4d, whereas anti-C4c mAb recognizes only C4b bound to the organism. Therefore, cofactor activity would result in decreased intact C4b detected on the organism (measured with the anti-C4c mAb), with a resultant increase in the C4d/C4c ratio. Consistent with their ability to bind C4bp, both MS11 as well as FA19 show higher C4d/C4c ratios. geo mean flu, geometric mean fluorescence.

Figure 1

Demonstration of cofactor activity on gonococcal strains that bind C4bp. Strains F62, MS11, and FA19 were incubated with 20% NHS for 30 min at 37°C, followed by detection of intact C4b fragments remaining on the bacterial surface by flow cytometry. C4bp cofactor activity would result in factor I–mediated cleavage of C4b to C4c and C4d. C4d remains bound to the bacterial surface, whereas the C4c fragment is released into solution. Anti-C4d mAb recognizes both C4b as well as C4d, whereas anti-C4c mAb recognizes only C4b bound to the organism. Therefore, cofactor activity would result in decreased intact C4b detected on the organism (measured with the anti-C4c mAb), with a resultant increase in the C4d/C4c ratio. Consistent with their ability to bind C4bp, both MS11 as well as FA19 show higher C4d/C4c ratios. geo mean flu, geometric mean fluorescence.

Figure 2

Por1A of FA19 and Por1B of MS11 are the acceptor molecules for C4bp. To demonstrate this for FA19 Por1A, we used pUNCH62 (reference 40) to replace F62 Por1B (C4bp nonbinder) with FA19 Por1A. The resultant strain, termed F62_PorFA19, bound C4bp in a flow cytometry assay when incubated with 10% NHS (left), indicating that FA19 Por1A was an acceptor molecule for C4bp. Similarly, using plasmid pUNCH61 (reference 40) we replaced F62 Por1B with MS11 Por1B, and observed that the isogenic mutant F62_PorMS11 bound C4bp (right), indicating that MS11 Por1B was an acceptor molecule for C4bp.

Figure 4

MS11 Por1B loops 5 and 7 together participate in forming a C4bp-binding region. Transformation of F62 with pUNCH61 (reference 40) resulted in strains with hybrid Por molecules F62_loop1MS11_loop2–8 and F62_loop1–4MS11_loop5–8. F62 sequence is indicated by black bars; MS11 sequence is represented by hatched bars. Both hybrids bound C4bp, suggesting that the C4bp binding region in MS11 lay in a region encompassed by loops 5–8. The BbsI-BsgI fragment of F62 was cloned into pUNCH61 to obtain pBUMC1 and this plasmid was used to transform F62. This yielded a gonococcal strain with hybrid Por molecule MS11_loop1 F62_loop2–7MS11_loop1–8, which did not bind C4bp, thus eliminating MS11 loop 8 as the C4bp binding loop. Using overlap extension PCR or site-directed mutagenesis on pBUMC1, we mutated loops 5, 6, or 7 either individually or in combination and then transformed F62. We noted that only hybrid Por molecules that contained both MS11 loop 5 and loop 7 bound C4bp. To further demonstrate the absolute requirement of MS11 loops 5 and 7, we mutated MS11 loops 5, 6, and 7 individually in pUNCH61 to resemble the corresponding F62 loop, and then transformed F62. Mutations of loop 5 (MS11_loop1–4F62_loop5MS11_loop6–8) and loop 7 (MS11_loop1–6F62_loop7MS11_loop8) resulted in complete abrogation of C4bp binding, whereas mutating loop 6 (MS11_loop1–5F62_loop6MS11_loop7–8) did not influence C4bp binding. Collectively, these data suggest that MS11 loops 5 and 7 together participate in forming a C4bp-binding domain.

Figure 3

FA19 Por1A loop 1 is required for C4bp binding. Transformation of F62 with pUNCH62 (containing the FA19 por1A gene) resulted in two classes of hybrid Por molecules, F62_loop1FA19_loop2–8 and F62_loop1–4FA19_loop2–8. F62 (Por1B) sequence is shown by solid black bars, and FA19 (Por1A) sequence by white bars. Neither class of hybrids bound C4bp. Loop 1 of FA19 Por1A is indicated by the gray shaded box; only parent strain FA19 that contains Por1A loop 1 binds C4bp, suggesting that loop 1 of Por1A is required for C4bp binding.

Figure 5

Enhanced IgM binding to F62_PorMS11: Por influences IgM binding to gonococci. Despite the ability of F62_PorMS11 to bind C4bp (see Fig. 2), the resulting transformant possessed the SS phenotype on bactericidal testing (100% killing in 10% NHS at 30 min). To determine the mechanism of serum sensitivity, we studied the binding of IgM, C3, C5b-9, and C4bp to F62_PorMS11 (gray shaded areas) in comparison with F62 (dotted lines) and F62_loop1MS11_loop2–8 (solid lines). In contrast to F62_PorMS11, F62_loop1MS11_loop2–8 was fully SR (100% survival in 10% NHS at 30 min), and differed from the former only at Por loop 1. F62_PorMS11 bound two- and fourfold more IgM than F62 and F62_loop1MS11_loop2–8, respectively. Levels of C3 and C5b-9 binding to F62_PorMS11 were intermediate compared with the two other strains. C4bp binding to F62_PorMS11 and F62_loop1MS11_loop2–8 were identical.

Figure 6

Characterization of Por–C4bp bonds. The influence of C4b, heparin and high-ionic strength on Por-C4bp binding was studied by flow cytometry. C4b, when added to C4bp in a 60-fold molar excess, inhibited C4bp binding to MS11 (Por1B) by 40% (A). 125 units of heparin completely inhibited C4bp binding to MS11 (B), and 575 mM NaCl reduced C4bp binding to MS11 by >80% (C). None of the above conditions influenced C4bp binding to FA19 (Por1A). To minimize bacterial lysis in these experiments, organisms were first fixed with 1% paraformaldehyde. Fixing gonococci before incubation with C4bp did not alter C4bp binding properties.

Figure 7

C4bp SCR 1 contains both Por1A as well as Por1B binding regions. (A) Mapping of Por binding sites in C4bp. Binding of recombinant C4bp (rC4bp) and seven rC4bp mutant molecules lacking individual SCRs to FA19 (Por1A) and MS11 (Por1B) were studied using flow cytometry. 10⁸ organisms were incubated with 2.5 μg of each rC4bp molecule, followed by detection with sheep polyclonal anti–human C4bp and disclosed with anti–sheep IgG-FITC. All rC4bp molecules that contained SCR 1 bound both FA19 as well as MS11; only the mutant that lacked SCR 1 (rC4bp ΔSCR1) did not bind either strain. Because artifactually lower fluorescence (threefold less binding compared with rC4bp) was observed when rC4bp ΔSCR6 was detected with the polyclonal anti-C4bp Ab, we used anti-C4bp mAb (Quidel Corp.) and mAb 67 (specific for SCR 4) to detect rC4bp ΔSCR6 bound to both strains. Compared with rC4bp, rC4bp ΔSCR6 bound with similar fluorescence intensities, when either mAb was used as a probe. Binding of rC4bp ΔSCR6 to both strains using the Quidel Corp. mAb is shown here (indicated by the asterisk). rC4bp ΔSCR1 did not bind to either strains when mAb 67 or the Quidel Corp. anti-C4bp mAb was used as the detection Ab. (B) Anti-C4bp SCR1 mAb blocks C4bp binding to gonococci. Further evidence that SCR 1 contains Por1A and Por1B binding regions was provided because mAb 104 that is specific for C4bp SCR 1 completely inhibits binding of C4bp to both FA19 (Por1A) as well as MS11 (Por1B; solid lines); control mAb 67 (specific for the irrelevant C4bp SCR 4) did not influence C4bp binding to either strain (gray shaded area) when compared with binding of pure C4bp alone (dashed lines).

Figure 7

C4bp SCR 1 contains both Por1A as well as Por1B binding regions. (A) Mapping of Por binding sites in C4bp. Binding of recombinant C4bp (rC4bp) and seven rC4bp mutant molecules lacking individual SCRs to FA19 (Por1A) and MS11 (Por1B) were studied using flow cytometry. 10⁸ organisms were incubated with 2.5 μg of each rC4bp molecule, followed by detection with sheep polyclonal anti–human C4bp and disclosed with anti–sheep IgG-FITC. All rC4bp molecules that contained SCR 1 bound both FA19 as well as MS11; only the mutant that lacked SCR 1 (rC4bp ΔSCR1) did not bind either strain. Because artifactually lower fluorescence (threefold less binding compared with rC4bp) was observed when rC4bp ΔSCR6 was detected with the polyclonal anti-C4bp Ab, we used anti-C4bp mAb (Quidel Corp.) and mAb 67 (specific for SCR 4) to detect rC4bp ΔSCR6 bound to both strains. Compared with rC4bp, rC4bp ΔSCR6 bound with similar fluorescence intensities, when either mAb was used as a probe. Binding of rC4bp ΔSCR6 to both strains using the Quidel Corp. mAb is shown here (indicated by the asterisk). rC4bp ΔSCR1 did not bind to either strains when mAb 67 or the Quidel Corp. anti-C4bp mAb was used as the detection Ab. (B) Anti-C4bp SCR1 mAb blocks C4bp binding to gonococci. Further evidence that SCR 1 contains Por1A and Por1B binding regions was provided because mAb 104 that is specific for C4bp SCR 1 completely inhibits binding of C4bp to both FA19 (Por1A) as well as MS11 (Por1B; solid lines); control mAb 67 (specific for the irrelevant C4bp SCR 4) did not influence C4bp binding to either strain (gray shaded area) when compared with binding of pure C4bp alone (dashed lines).

Figure 7

C4bp SCR 1 contains both Por1A as well as Por1B binding regions. (A) Mapping of Por binding sites in C4bp. Binding of recombinant C4bp (rC4bp) and seven rC4bp mutant molecules lacking individual SCRs to FA19 (Por1A) and MS11 (Por1B) were studied using flow cytometry. 10⁸ organisms were incubated with 2.5 μg of each rC4bp molecule, followed by detection with sheep polyclonal anti–human C4bp and disclosed with anti–sheep IgG-FITC. All rC4bp molecules that contained SCR 1 bound both FA19 as well as MS11; only the mutant that lacked SCR 1 (rC4bp ΔSCR1) did not bind either strain. Because artifactually lower fluorescence (threefold less binding compared with rC4bp) was observed when rC4bp ΔSCR6 was detected with the polyclonal anti-C4bp Ab, we used anti-C4bp mAb (Quidel Corp.) and mAb 67 (specific for SCR 4) to detect rC4bp ΔSCR6 bound to both strains. Compared with rC4bp, rC4bp ΔSCR6 bound with similar fluorescence intensities, when either mAb was used as a probe. Binding of rC4bp ΔSCR6 to both strains using the Quidel Corp. mAb is shown here (indicated by the asterisk). rC4bp ΔSCR1 did not bind to either strains when mAb 67 or the Quidel Corp. anti-C4bp mAb was used as the detection Ab. (B) Anti-C4bp SCR1 mAb blocks C4bp binding to gonococci. Further evidence that SCR 1 contains Por1A and Por1B binding regions was provided because mAb 104 that is specific for C4bp SCR 1 completely inhibits binding of C4bp to both FA19 (Por1A) as well as MS11 (Por1B; solid lines); control mAb 67 (specific for the irrelevant C4bp SCR 4) did not influence C4bp binding to either strain (gray shaded area) when compared with binding of pure C4bp alone (dashed lines).

Figure 8

Functional effects of blocking C4bp binding to gonococci. (A) fAb 104 inhibits C4bp binding to gonococci. 20 μg of fAb fragments generated from mAb 104, when added to 10% NHS, inhibited C4bp binding to FA19 and MS11 equally (gray shaded area). C4bp binding in the presence of NHS alone is shown by the solid line. (B) Diverting C4bp from the bacterial surface converts SR gonococci to an SS phenotype. A serum bactericidal assay was performed to assess the functional effects of inhibiting the binding of C4bp binding to the bacteria. FA19 and MS11 were incubated either with 10% NHS alone, 10% NHS plus 25 μg fAb 104, or 10% NHS plus 25 μg fAb 67 (negative control) for 30 min at 37°C. fAb 104 abrogated C4bp binding to the bacterial surface resulting in 100% killing of FA19 and 80% killing of MS11. NHS alone or NHS with (irrelevant) fAb 67 resulted in no significant killing of either strain. (C) Kinetics of bacterial killing by an unimpeded classical pathway. Abrogation of C4bp binding to the bacterial surface resulted in slow, sustained bacterial killing, with an almost linear decrease in bacterial viability over time of both FA19 (solid line) and MS11 (dotted line).

Figure 8

Functional effects of blocking C4bp binding to gonococci. (A) fAb 104 inhibits C4bp binding to gonococci. 20 μg of fAb fragments generated from mAb 104, when added to 10% NHS, inhibited C4bp binding to FA19 and MS11 equally (gray shaded area). C4bp binding in the presence of NHS alone is shown by the solid line. (B) Diverting C4bp from the bacterial surface converts SR gonococci to an SS phenotype. A serum bactericidal assay was performed to assess the functional effects of inhibiting the binding of C4bp binding to the bacteria. FA19 and MS11 were incubated either with 10% NHS alone, 10% NHS plus 25 μg fAb 104, or 10% NHS plus 25 μg fAb 67 (negative control) for 30 min at 37°C. fAb 104 abrogated C4bp binding to the bacterial surface resulting in 100% killing of FA19 and 80% killing of MS11. NHS alone or NHS with (irrelevant) fAb 67 resulted in no significant killing of either strain. (C) Kinetics of bacterial killing by an unimpeded classical pathway. Abrogation of C4bp binding to the bacterial surface resulted in slow, sustained bacterial killing, with an almost linear decrease in bacterial viability over time of both FA19 (solid line) and MS11 (dotted line).

Figure 8

Functional effects of blocking C4bp binding to gonococci. (A) fAb 104 inhibits C4bp binding to gonococci. 20 μg of fAb fragments generated from mAb 104, when added to 10% NHS, inhibited C4bp binding to FA19 and MS11 equally (gray shaded area). C4bp binding in the presence of NHS alone is shown by the solid line. (B) Diverting C4bp from the bacterial surface converts SR gonococci to an SS phenotype. A serum bactericidal assay was performed to assess the functional effects of inhibiting the binding of C4bp binding to the bacteria. FA19 and MS11 were incubated either with 10% NHS alone, 10% NHS plus 25 μg fAb 104, or 10% NHS plus 25 μg fAb 67 (negative control) for 30 min at 37°C. fAb 104 abrogated C4bp binding to the bacterial surface resulting in 100% killing of FA19 and 80% killing of MS11. NHS alone or NHS with (irrelevant) fAb 67 resulted in no significant killing of either strain. (C) Kinetics of bacterial killing by an unimpeded classical pathway. Abrogation of C4bp binding to the bacterial surface resulted in slow, sustained bacterial killing, with an almost linear decrease in bacterial viability over time of both FA19 (solid line) and MS11 (dotted line).