Inhibition of the alternative pathway of nonhuman infant complement by porin B2 contributes to virulence of Neisseria meningitidis in the infant rat model

Abstract

Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats.

FIG 1

fH-dependent regulation of the AP of baby rabbit complement by PorB2-expressing N. meningitidis. (A and B) Decreased baby rabbit C3 fragment deposition on PorB2-expressing meningococci. Seven strains of N. meningitidis bacteria that lacked capsular polysaccharide, LOS sialic acid, fHbp, and NspA expression (quadruple mutants) were screened for surface C3 deposition when incubated with baby rabbit complement (20% [vol/vol]) containing 10 mM Mg-EGTA to block classical and lectin pathway activity and permit only AP activation. Histograms from a representative experiment are shown in panel A. The percentage of positive events for baby rabbit C3 fragment deposition relative to control organisms incubated with heat-inactivated serum (y axis) is shown in panel B (gated to include 5% of events as positive [+ve] in the control histogram). Each bar represents the mean (standard error of the mean) of seven independent experiments. *, P < 0.05 (one-way ANOVA with Tukey's posttest for pairwise comparisons). −ve, negative. (C) Blocking the human fH binding site on PorB2 with human fH67/Fc enhances baby rabbit C3 deposition and provides evidence for function of rabbit fH on PorB2-expressing meningococci. Quadruple mutants of strain 4243 (PorB2) and H44/76 (PorB3; control) were incubated with either buffer alone (−), human fH67/Fc (67), or human fH18–20/Fc (18–20; negative-control protein) at concentrations of 10 μg/ml, followed by the addition of Mg-EGTA-treated baby rabbit complement (20% [vol/vol]) (left graph). C3 deposited on bacteria was detected by FACS. Similar experiments, performed with Mg-EGTA-treated human serum (right graph), demonstrate the functional interaction of human fH with PorB2 on the quadruple mutant of 4243. The y axis shows the percentage of positive events relative to controls treated with heat-inactivated complement. Bars show the means (standard error of the mean) of at least three separate experiments. ***, P < 0.001, compared to reaction mixtures containing buffer alone or fH18–20/Fc.

FIG 2

PorB2-expressing quadruple mutants inhibit the AP of infant rat complement. (A) Seven N. meningitidis quadruple mutants were incubated with 10% (vol/vol) Mg-EGTA-treated pooled serum from 8- or 9-day-old infant rats, and rat C3 fragments deposited on bacteria were measured by FACS. Representative histogram tracings (one of three reproducible repeats) are shown. (B) Data are from three separate experiments with infant rat serum, expressed as a percentage of positive events relative to heat-inactivated serum controls (gated to yield 5% of positive events [+ve] in the negative-control sample as shown). Each bar shows the mean (standard error of the mean). *, P < 0.05 (one-way ANOVA with Tukey's posttest for pairwise comparisons).

FIG 3

Isogenic mutants of N. meningitidis that express PorB2 inhibit the rat AP and resist killing by infant serum. Isogenic mutant strains that differed only in their PorB molecule were created in the background of strain H44/76 fHbp nspA. Both mutants lacked fHbp and NspA but expressed capsular polysaccharide and LOS sialic acid. (A) PorB2 expression regulates infant rat AP. Bacteria were incubated with Mg-EGTA-treated infant rat serum (final concentration, either 20% or 40%), and deposited rat C3 was measured by FACS. Gray bars, H44/76 fHbp nspA expressing PorB3; black bars, H44/76 fHbp nspA expressing PorB2; open bars, control (no serum added). Each bar shows the percentage of positive events relative to the controls (set at 5%), and data represent the means (standard error of the mean) of three independent experiments. ***, P < 0.001. (B) PorB2 expression increases resistance to bacteriolysis in infant rat serum. Serum bactericidal assays with the isogenic PorB mutants (described in panel A) were performed with infant rat serum. Each bar represents the mean (standard error of the mean) of three independent experiments. **, P ≤ 0.002.

FIG 4

Expression of PorB2 enhances bacteremia in the infant rat model. Six- to 8-day-old WT infant Wistar rats (A) or human fH transgenic Wistar rats (B) were challenged intraperitoneally with either H44/76 fHbp nspA-PorB3 (B3) or its isogenic mutant, H44/76 fHbp nspA-PorB2 (B2). Both mutants expressed capsule and LOS sialic acid. Bacterial inocula used for each experiment are indicated alongside each graph (upper left). Colony-forming units (CFU)/ml in blood of individual animals at 6 h are indicated on the y axis. ****, P < 0.0001. When the data from both challenge doses were analyzed together, the proportion of positive blood cultures in the animals challenged with the PorB3 mutant was lower than that of animals challenged with the PorB2 mutant.