Factor H-dependent alternative pathway inhibition mediated by porin B contributes to virulence of Neisseria meningitidis

Abstract

The identification of "factor H binding protein (fHbp)-null" invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines.

Importance: The widespread use of antimeningococcal vaccines based on factor H (fH) binding protein (fHbp) is imminent. Meningococci that lack fHbp were recently isolated from persons with invasive disease, and these fHbp-null strains could spawn vaccine failure. Our report provides a molecular basis for an explanation of how fHbp-null strains may evade the host immune system. Meningococci possess several mechanisms to subvert killing by the alternative pathway (AP) of complement, including production of the fHbp and NspA fH binding proteins. Here we show that a meningococcal protein called porin B2 (PorB2) contributes to inhibition of the AP on the bacterial surface. A majority of the "fHbp-null" isolates identified, as well as all members of a "hypervirulent" lineage (called ST-11), express PorB2. Our findings highlight the potential for the emergence of fHbp-negative strains that are able to regulate the AP and may be associated with fHbp vaccine failure.

FIG 1

Regulation of the human AP by PorB2-expressing N. meningitidis. Seven strains of N. meningitidis that lacked capsular polysaccharide, LOS sialic acid, and fHbp and NspA expression (quadruple mutants) were screened for AP-mediated C3 deposition with NHS-Mg/EGTA (20% [vol/vol]). (A) C3 deposition measured by FACS analysis. The percentage of positive events relative to control organisms incubated with heat-inactivated serum (gated to yield 5% of positive events in the negative-control sample as shown in Fig. 1B [+ve]) is represented on the y axis. Each bar represents the standard error of the mean (SEM) of the results of three independent experiments. ***, P < 0.001 (1-way ANOVA with Tukey’s posttest for pairwise comparisons). (B) Histograms from an experiment representative of those described for panel A; data represent the median fluorescence of C3 binding of the entire bacterial population. (C) AP regulation by PorB2-expressing strains revealed using purified AP components. Quadruple mutants were incubated with purified C3 (500 µg/ml), factor B (100 µg/ml), factor D (2 µg/ml), and factor H (100 µg/ml). C3b deposited on bacteria was measured by FACS analysis. Controls were bacteria incubated with C3 alone. The amount of deposited C3b is represented as median fluorescence (y axis). Each bar represents the mean (range) of the results of two separate experiments. **, P < 0.01. (D) 4243 (PorB2) activates the AP at a lower rate than H44/76 (PorB3). Quadruple mutants of 4243 (PorB2) and H44/76 (PorB3) were incubated with NHS-Mg/EGTA. Supernatants collected at the times indicated (x axis) were assayed for C3a by enzyme-linked immunosorbent assay (ELISA). Each datum point represents the SEM of the results of three independent experiments. **, P < 0.01 (2-way ANOVA).

FIG 2

Human fH inhibits C3 deposition by adult nonhuman complement on PorB2-expressing quadruple mutants. Quadruple mutants of 7 strains of N. meningitidis were incubated with adult rabbit complement (A) or adult mouse serum (B) in either the absence (“−”; solid black bars) or the presence (“+”; gray bars) of human fH (80 µg/ml), and levels of C3 fragments deposited on bacteria were measured by FACS analysis. The control (open bar) represents C3 deposition when bacteria were incubated with heat-inactivated complement. The percentage of positive events relative to controls (bacteria plus heat-inactivated complement) is shown on the y axis. Each bar represents the SEM of the results of 2 to 3 independent observations. **, P < 0.01; ***, P < 0.001 (1-way ANOVA).

FIG 3

PorB2 mediates regulation of the human AP. Isogenic strains that differed only in PorB expression (either PorB3_H44/76 or PorB2₄₂₄₃) were created in H44/76. All mutants lacked fHbp and NspA and were either unencapsulated with no LOS sialic acid (“quadruple”; “Cap−”) or possessed a capsule and LOS acid (H44/76 fHbp nspA-PorB3 and H44/76 fHbp nspA-PorB2; “Cap+”). Bacteria were incubated with NHS-Mg/EGTA (20% [vol/vol]), and C3 deposited on the bacterial surface was measured by FACS analysis. (A and C) Representative histogram tracings of C3 deposition on the unencapsulated (Cap-) and encapsulated (Cap+) strains. Bacteria incubated with heat-inactivated serum served as negative controls (dashed lines). (B and D) Quantitative representation (percentage of positive events on the y axis; mean [SEM] of the results of three independent experiments) of C3 deposition on the Cap- and Cap+ isogenic PorB mutants, respectively. The percentage of positive events relative to control organisms incubated with heat-inactivated serum (gated to yield 5% of positive events in the negative-control sample as shown in Fig. 1B [+ve]) is represented on the y axis. Wild-type parent strains H44/76 and 4243 are shown as comparators. Note the different y-axis scales in panels B and D. ***, P < 0.001; **, P < 0.01.

FIG 4

PorB2 binds full-length fH, fH domains 6 and 7, and FHL-1. (A and B) Binding of human fH to quadruple mutants of H44/76 that express either PorB3_44/76 (H44/76-PorB3) or PorB2₄₂₄₃ (H44/76-PorB2) (A) and quadruple mutants of N. meningitidis strains that express either PorB3 (A2594, H44/76, and NZ98/254) or PorB2 (C2120, W171, 4243, and 2996) (B). Quadruple mutants were incubated with purified fH (100 µg/ml), and bound fH was measured by FACS analysis (graphs labeled “no cross-linker”). In some experiments, bound fH was cross-linked to the surface with paraformaldehyde (final concentration, 1% [vol/vol]) prior to detection by FACS analysis (graphs labeled “paraformaldehyde cross-linker”). The negative control (control) was a reaction mixture that lacked fH (neg. control). Binding of fH to wild-type H44/76 that expresses fHbp, NspA, capsule, and LOS sialic acid is shown as a positive control in panel B (pos. control). The amount of bound fH is represented on the y axis as fold change in median fluorescence relative to the H44/76 quadruple mutant that expresses PorB3 (in panel A) or the H44/76 quadruple mutant (in panel B) with paraformaldehyde cross-linker. Each bar represents the SEM of the results of 2 to 3 independent experiments (note that some error bars are small and are not easily seen on the figure). **, P < 0.01; *, P < 0.05 (1-way ANOVA with Tukey’s posttest for pairwise comparisons). (C) PorB2₄₂₄₃ binds to fH67/Fc. Binding of human fH67/Fc to quadruple mutants of H44/76 (blue line), 4243 (green line), and H44/76 that express either PorB3_44/76 (H44/76-PorB3; red line) or PorB2₄₂₄₃ (H44/76-PorB2; gray shading) was measured by FACS analysis. The negative control (broken line) was a reaction mixture that lacked fH67/Fc. x axis, fluorescence on a log₁₀ scale; y axis, counts. Data from an experiment representative of at least two reproducible repeats are shown. (D) A quadruple mutant of strain 4243 binds FHL-1. Binding of FHL-1 (10 µg/ml) to the 4243 quadruple mutant (gray-shaded histogram) was measured by FACS analysis using polyclonal goat anti-human fH. FHL-1 was omitted in the negative control (broken line). The positive control shows binding of FHL-1 to wild-type H44/76 (solid black line). Numbers alongside histograms represent the median fluorescence, and the fill/outline corresponds to that of the histograms.

FIG 5

PorB2 expression enhances serum resistance. Data represent percent survival of isogenic H44/76 fHbp nspA expressing either PorB2₄₂₄₃ (PorB2; open bar) or PorB3_H44/76 (PorB3; gray bar) in 20% absorbed serum that contained anti-group B capsule antibody, SEAM 12, at the concentrations indicated. Heat-inactivated (HI) sera and NHS without added SEAM 12 (0 µg/ml) served as controls. Percent survival relative to time 0 is plotted on the y axis. Each bar represents the SEM of the results of three independent observations. **, P < 0.01; ****, P < 0.0001 (2-way ANOVA).