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Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility.

Caesar JJ, Lavender H, Ward PN, Exley RM, Eaton J, Chittock E, Malik TH, Goiecoechea De Jorge E, Pickering MC, Tang CM, Lea SM - Elife (2014)

Bottom Line: We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH.Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone.The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.

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Characterisation of the novel anti-CFHR3 antibody HSL1.(A) Western blotting of recombinant CFHR3 and sera from individuals sufficient or deficient in CFHR3 demonstrates that HSL1 is specific for CFHR3. (B) For screening of mAbs, ELISA plates (F96 maxisorp, Nunc) were coated with C-terminal pair of CCP domains of CFH and the CFHRs (5 µg/ml, 50 µl per well) overnight at room temperature before blocking with 2% skimmed milk in PBS 0.05% Tween 20. Plates were incubated with neat hybridoma culture supernatant and binding was detected with goat anti-mouse HRP (1 in 5000, Dako) followed by ELISA substrate and stop solution (Roche).DOI:http://dx.doi.org/10.7554/eLife.04008.007
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fig3s1: Characterisation of the novel anti-CFHR3 antibody HSL1.(A) Western blotting of recombinant CFHR3 and sera from individuals sufficient or deficient in CFHR3 demonstrates that HSL1 is specific for CFHR3. (B) For screening of mAbs, ELISA plates (F96 maxisorp, Nunc) were coated with C-terminal pair of CCP domains of CFH and the CFHRs (5 µg/ml, 50 µl per well) overnight at room temperature before blocking with 2% skimmed milk in PBS 0.05% Tween 20. Plates were incubated with neat hybridoma culture supernatant and binding was detected with goat anti-mouse HRP (1 in 5000, Dako) followed by ELISA substrate and stop solution (Roche).DOI:http://dx.doi.org/10.7554/eLife.04008.007

Mentions: Next we investigated whether CFHR3 binds to the surface of N. meningitidis. We generated a CFHR3-specific monoclonal antibody (mAb) (Figure 3—figure supplement 1), designated HSL1. By flow cytometry we demonstrated that serum CFHR3 bound N. meningitidis M1239 (which expresses V3.28 fHbp) in a fHbp-dependent manner. No binding was detected following incubation of bacteria in sera from an individual homozygous for an allele in which there is combined deletion of the CFHR1 and CFHR3 genes (ΔCFHR3/1) (ΔCFHR3/1, Figure 3A,B,C). We therefore tested the functional consequences of CFHR3 binding by comparing the effect of adding full length or domains of CFHR3 to N. meningitidis prior to incubation in normal human serum (NHS). Addition of full-length CFHR3 or CFHR312, which contains the fHbp binding site (Figure 1C) but has no CFH antagonist activity (Figure 1B), resulted in significantly reduced bacterial survival (CFHR3 51%; CFHR312 70%; no protein or CFHR345 83%, Figure 3D); in contrast, CFHR3 had no detectable effect in the presence of heat-inactivated NHS (which lacks complement activity, Figure 3D). Full-length CFHR3 has a more marked effect on bacterial survival than CFHR312, consistent with the ability of the full-length CHHR3 to bind to C3b deposited on the bacterial surface (via CFHR345) as well as competing with CFH for fHbp (via CFHR312). As CFHR345 does not bind fHbp (Figure 1C), its CFH antagonist activity is not effectively localized to the meningococcal surface, providing an explanation for why these domains have no effect on bacterial survival (Figure 3D). Similarly, addition of CFHR3 has no effect on serum survival of bacteria that do not express any CFH-binding protein (Figure 3—figure supplement 2A, N. meningitidis strain lacking fHbp expression; Figure 3—figure supplement 2B, Escherichia coli strain with no expression of any CFH-binding protein).10.7554/eLife.04008.006Figure 3.N. meningitidis binds CFHR3 on its surface in an fHbp-dependent manner promoting complement-mediated lysis.


Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility.

Caesar JJ, Lavender H, Ward PN, Exley RM, Eaton J, Chittock E, Malik TH, Goiecoechea De Jorge E, Pickering MC, Tang CM, Lea SM - Elife (2014)

Characterisation of the novel anti-CFHR3 antibody HSL1.(A) Western blotting of recombinant CFHR3 and sera from individuals sufficient or deficient in CFHR3 demonstrates that HSL1 is specific for CFHR3. (B) For screening of mAbs, ELISA plates (F96 maxisorp, Nunc) were coated with C-terminal pair of CCP domains of CFH and the CFHRs (5 µg/ml, 50 µl per well) overnight at room temperature before blocking with 2% skimmed milk in PBS 0.05% Tween 20. Plates were incubated with neat hybridoma culture supernatant and binding was detected with goat anti-mouse HRP (1 in 5000, Dako) followed by ELISA substrate and stop solution (Roche).DOI:http://dx.doi.org/10.7554/eLife.04008.007
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273445&req=5

fig3s1: Characterisation of the novel anti-CFHR3 antibody HSL1.(A) Western blotting of recombinant CFHR3 and sera from individuals sufficient or deficient in CFHR3 demonstrates that HSL1 is specific for CFHR3. (B) For screening of mAbs, ELISA plates (F96 maxisorp, Nunc) were coated with C-terminal pair of CCP domains of CFH and the CFHRs (5 µg/ml, 50 µl per well) overnight at room temperature before blocking with 2% skimmed milk in PBS 0.05% Tween 20. Plates were incubated with neat hybridoma culture supernatant and binding was detected with goat anti-mouse HRP (1 in 5000, Dako) followed by ELISA substrate and stop solution (Roche).DOI:http://dx.doi.org/10.7554/eLife.04008.007
Mentions: Next we investigated whether CFHR3 binds to the surface of N. meningitidis. We generated a CFHR3-specific monoclonal antibody (mAb) (Figure 3—figure supplement 1), designated HSL1. By flow cytometry we demonstrated that serum CFHR3 bound N. meningitidis M1239 (which expresses V3.28 fHbp) in a fHbp-dependent manner. No binding was detected following incubation of bacteria in sera from an individual homozygous for an allele in which there is combined deletion of the CFHR1 and CFHR3 genes (ΔCFHR3/1) (ΔCFHR3/1, Figure 3A,B,C). We therefore tested the functional consequences of CFHR3 binding by comparing the effect of adding full length or domains of CFHR3 to N. meningitidis prior to incubation in normal human serum (NHS). Addition of full-length CFHR3 or CFHR312, which contains the fHbp binding site (Figure 1C) but has no CFH antagonist activity (Figure 1B), resulted in significantly reduced bacterial survival (CFHR3 51%; CFHR312 70%; no protein or CFHR345 83%, Figure 3D); in contrast, CFHR3 had no detectable effect in the presence of heat-inactivated NHS (which lacks complement activity, Figure 3D). Full-length CFHR3 has a more marked effect on bacterial survival than CFHR312, consistent with the ability of the full-length CHHR3 to bind to C3b deposited on the bacterial surface (via CFHR345) as well as competing with CFH for fHbp (via CFHR312). As CFHR345 does not bind fHbp (Figure 1C), its CFH antagonist activity is not effectively localized to the meningococcal surface, providing an explanation for why these domains have no effect on bacterial survival (Figure 3D). Similarly, addition of CFHR3 has no effect on serum survival of bacteria that do not express any CFH-binding protein (Figure 3—figure supplement 2A, N. meningitidis strain lacking fHbp expression; Figure 3—figure supplement 2B, Escherichia coli strain with no expression of any CFH-binding protein).10.7554/eLife.04008.006Figure 3.N. meningitidis binds CFHR3 on its surface in an fHbp-dependent manner promoting complement-mediated lysis.

Bottom Line: We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH.Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone.The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.

Show MeSH
Related in: MedlinePlus