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Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Bidmos FA, Chan H, Praekelt U, Tauseef I, Ali YM, Kaczmarski EB, Feavers I, Bayliss CD - PLoS ONE (2015)

Bottom Line: The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease.These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood.These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

No MeSH data available.


Related in: MedlinePlus

Testing the serum bactericidal assay of anti-HmbR antisera.Meningococcal cell suspensions, grown in iron-replete (uninduced) and iron-deficient (induced) conditions, and assays were performed as described for Fig 7. Antisera/antibodies were tested at the indicated dilutions for each sera against the indicated strains:- (A) polyclonal anti-rHmbR antisera, blue line, uninduced MC58, red line induced MC58; (B) pooled anti-HmbR mAbs, blue line induced MC58, red line induced MC58ΔHmbR; (C) a PorA monoclonal antibody, P1.7, blue line induced MC58, red line induced MC58ΔHmbR. Inoc, cell count after 0 minutes; No Ab, cell count after 60 minutes incubation in serum alone. Graphs show mean values for two independent experiments for the pooled anti-rHmbR antisera and one for the PorA P1.7 and anti-HmbR mAbs.
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pone.0133855.g008: Testing the serum bactericidal assay of anti-HmbR antisera.Meningococcal cell suspensions, grown in iron-replete (uninduced) and iron-deficient (induced) conditions, and assays were performed as described for Fig 7. Antisera/antibodies were tested at the indicated dilutions for each sera against the indicated strains:- (A) polyclonal anti-rHmbR antisera, blue line, uninduced MC58, red line induced MC58; (B) pooled anti-HmbR mAbs, blue line induced MC58, red line induced MC58ΔHmbR; (C) a PorA monoclonal antibody, P1.7, blue line induced MC58, red line induced MC58ΔHmbR. Inoc, cell count after 0 minutes; No Ab, cell count after 60 minutes incubation in serum alone. Graphs show mean values for two independent experiments for the pooled anti-rHmbR antisera and one for the PorA P1.7 and anti-HmbR mAbs.

Mentions: For the anti-HpuA hSBAs, a 5% human serum extract was used due to the high sensitivity of strain 8047 to human serum. The anti-r8047-HpuA serum exhibited no bactericidal activity against either strain 8047 wt and 8047ΔhpuAB (Fig 7A). Contrastingly, a P1.2 mAb specific for VR2 of PorA exhibited high levels of bactericidal activity for both strains with a titre of ≥ 640 (Fig 7B). The bactericidal activity of monoclonal and polyclonal anti-HmbR antibodies was performed using strain MC58, mutants thereof and 20% serum. Bactericidal activity was observed with an anti-PorA P1.7 mAb (hSBA titre of ≥ 320) but not with pooled anti-HmbR mAbs or anti-rMC58-HmbR antisera (Fig 8).


Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Bidmos FA, Chan H, Praekelt U, Tauseef I, Ali YM, Kaczmarski EB, Feavers I, Bayliss CD - PLoS ONE (2015)

Testing the serum bactericidal assay of anti-HmbR antisera.Meningococcal cell suspensions, grown in iron-replete (uninduced) and iron-deficient (induced) conditions, and assays were performed as described for Fig 7. Antisera/antibodies were tested at the indicated dilutions for each sera against the indicated strains:- (A) polyclonal anti-rHmbR antisera, blue line, uninduced MC58, red line induced MC58; (B) pooled anti-HmbR mAbs, blue line induced MC58, red line induced MC58ΔHmbR; (C) a PorA monoclonal antibody, P1.7, blue line induced MC58, red line induced MC58ΔHmbR. Inoc, cell count after 0 minutes; No Ab, cell count after 60 minutes incubation in serum alone. Graphs show mean values for two independent experiments for the pooled anti-rHmbR antisera and one for the PorA P1.7 and anti-HmbR mAbs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514712&req=5

pone.0133855.g008: Testing the serum bactericidal assay of anti-HmbR antisera.Meningococcal cell suspensions, grown in iron-replete (uninduced) and iron-deficient (induced) conditions, and assays were performed as described for Fig 7. Antisera/antibodies were tested at the indicated dilutions for each sera against the indicated strains:- (A) polyclonal anti-rHmbR antisera, blue line, uninduced MC58, red line induced MC58; (B) pooled anti-HmbR mAbs, blue line induced MC58, red line induced MC58ΔHmbR; (C) a PorA monoclonal antibody, P1.7, blue line induced MC58, red line induced MC58ΔHmbR. Inoc, cell count after 0 minutes; No Ab, cell count after 60 minutes incubation in serum alone. Graphs show mean values for two independent experiments for the pooled anti-rHmbR antisera and one for the PorA P1.7 and anti-HmbR mAbs.
Mentions: For the anti-HpuA hSBAs, a 5% human serum extract was used due to the high sensitivity of strain 8047 to human serum. The anti-r8047-HpuA serum exhibited no bactericidal activity against either strain 8047 wt and 8047ΔhpuAB (Fig 7A). Contrastingly, a P1.2 mAb specific for VR2 of PorA exhibited high levels of bactericidal activity for both strains with a titre of ≥ 640 (Fig 7B). The bactericidal activity of monoclonal and polyclonal anti-HmbR antibodies was performed using strain MC58, mutants thereof and 20% serum. Bactericidal activity was observed with an anti-PorA P1.7 mAb (hSBA titre of ≥ 320) but not with pooled anti-HmbR mAbs or anti-rMC58-HmbR antisera (Fig 8).

Bottom Line: The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease.These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood.These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

No MeSH data available.


Related in: MedlinePlus