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Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in
Kathleen Y Dunphy1, Peter T Beernink, Barbara Brogioni
1Center for Immunobiology and Vaccine Development, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Abstract:
Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHbp from an epidemic Norwegian strain (ID 1). By surface plasmon resonance, association rate constants (k(a)) for fHbp ID 14 and 15 were similar to those for ID 1, but dissociation rate constants (k(d)) were 4- to 10-fold higher than those for ID 1. To determine the effect of fH affinity on fHbp fitness, we prepared isogenic mutants of strain H44/76 that expressed fHbp ID 1, 14, or 15. By flow cytometry, mutants expressing fHbp ID 14 or 15 had lower fH binding than ID 1. When incubated in plasma or blood of nonimmune donors, all three mutants showed similar increases in CFU/ml. In contrast, an isogenic fHbp knockout mutant, which grew well in broth, was rapidly killed in plasma or blood. Thus, although fHbp expression was required for survival of strain H44/76 in blood or plasma, expression of two natural fHbp sequence variants with lower fH affinity had minimal or no effect on nonimmune clearance. One reason may be the high fH concentrations in normal serum, which favor saturation of fH binding to fHbp, even when dissociation rates varied over 10-fold.
Insights
Factor H (fH) binding to Neisseria meningitidis fHbp is crucial for immune evasion. Variants with lower fH binding affinity did not impair bacterial survival in human blood, suggesting high serum fH levels compensate.
Area of Science:
- Microbiology
- Immunology
- Bacterial Pathogenesis
Background:
- Neisseria meningitidis evades host defenses by binding complement inhibitor factor H (fH).
- Factor H-binding protein (fHbp) is a key meningococcal surface protein and an fH ligand.
- Understanding fHbp variants' interaction with fH is critical for vaccine development.
Purpose of the Study:
- To investigate the impact of natural sequence variations in fHbp on fH binding affinity.
- To assess how altered fH binding affects meningococcal survival in human blood and plasma.
Main Methods:
- Recombinant fHbp variants were generated and characterized for fH binding using ELISA and surface plasmon resonance.
- Isogenic Neisseria meningitidis mutants expressing different fHbp variants were created.
- Bacterial survival was quantified by CFU/ml after incubation in human blood and plasma; fH binding was assessed by flow cytometry.
Main Results:
- Two natural fHbp variants (ID 14 and 15) exhibited 4- to 10-fold higher dissociation rates from fH compared to a reference (ID 1), indicating lower binding affinity.
- Mutants expressing fHbp variants with lower fH affinity showed reduced fH binding on their surface.
- Despite reduced fH binding, all fHbp-expressing mutants demonstrated similar survival in nonimmune human blood and plasma, unlike the fHbp knockout mutant.
Conclusions:
- fHbp expression is essential for Neisseria meningitidis survival in human blood and plasma.
- Natural fHbp variants with significantly lower fH binding affinity do not impair bacterial clearance in nonimmune individuals.
- High concentrations of factor H in serum may lead to saturation of fHbp binding sites, masking the functional impact of reduced affinity variants.
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