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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

Reactivity of anti-rHpuA and anti-rHpuB antisera with HpuAB in meningococcal lysates.Meningococcal cells of strain 8047, an isogenic Δhpu mutant and two phase variants of a carriage strain (N88.1, hpu-OFF; and N272.1, hpu-ON) were grown to mid-log (OD600 = ~0.5) before 30 μM of desferal was added to produce iron-limited conditions. Cultures were also grown concurrently in iron-replete conditions. All cultures were incubated for two hours before heat-inactivation at 56°C overnight. Lysates were prepared using an equal number of OD units and subject to SDS-PAGE electrophoresis and Western blotting. Blots were then probed with 1:500 dilutions of mouse polyclonal sera followed by a 1:2000 dilution of an anti-mouse IgG HRP-conjugate:- upper panel, anti-rN88-HpuA; middle panel, anti-r8047-HpuA; lower panel anti-r8047-HpuB. Lysates were from:- strain N88.1, induced (lane 1); 8047ΔhpuAB, induced (lane 2); wild-type 8047, uninduced (lane 3); wild-type 8047, induced (lane 4); N272.1 uninduced (lane 5); and N272.1 induced (lane 6).
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pone.0133855.g003: Reactivity of anti-rHpuA and anti-rHpuB antisera with HpuAB in meningococcal lysates.Meningococcal cells of strain 8047, an isogenic Δhpu mutant and two phase variants of a carriage strain (N88.1, hpu-OFF; and N272.1, hpu-ON) were grown to mid-log (OD600 = ~0.5) before 30 μM of desferal was added to produce iron-limited conditions. Cultures were also grown concurrently in iron-replete conditions. All cultures were incubated for two hours before heat-inactivation at 56°C overnight. Lysates were prepared using an equal number of OD units and subject to SDS-PAGE electrophoresis and Western blotting. Blots were then probed with 1:500 dilutions of mouse polyclonal sera followed by a 1:2000 dilution of an anti-mouse IgG HRP-conjugate:- upper panel, anti-rN88-HpuA; middle panel, anti-r8047-HpuA; lower panel anti-r8047-HpuB. Lysates were from:- strain N88.1, induced (lane 1); 8047ΔhpuAB, induced (lane 2); wild-type 8047, uninduced (lane 3); wild-type 8047, induced (lane 4); N272.1 uninduced (lane 5); and N272.1 induced (lane 6).

Mentions: Both anti-rHpuA mouse antisera bound to a protein of the expected size (~40-kDa) in Western blots of meningococcal lysates of strain 8047 and CC174 hpuAB-ON variant (N272.1) but not the 8047ΔhpuAB mutant or the CC174 hpuAB-OFF variant (N88.1), indicating specific reactivity with HpuA (Fig 3). Surface expression of HpuA in the 8047 and N272.1 strains was demonstrated by detection of high reactivity to formalin-fixed cells in the FACs assays (Fig 4A and 4B). No difference was observed, by Western blotting or FACs, in binding of anti-r8047-HpuA antibodies to strains N272.1 and 8047 whereas the anti-rN88-HpuA sera exhibited a major reduction in reactivity with 8047-HpuA as compared to N88.1-HpuA. This suggests that there are either differences in the level of HpuA expression, with a lower level of expression in strain 8047 as compared to the CC174 strain, or differences in the amounts of variant specific antibodies in each serum (i.e. more variant-specific antibodies in the N88 versus the 8047 sera). Further testing of the reactivity of anti-HpuA antisera to a panel of carriage strains in colony immunoblots showed specific reactivity of anti-rN88-HpuA to only the cognate CC-174 strains, N46 and N52 (Fig 4E). These CC-174 isolates were isolated from the same hall of residence and represent a cluster of genotypically identical strains that are thought to be a product of clonal expansion within the hall of residence [2]. A very weak interaction between anti-r8047-HpuA antibodies and the CC-174 strains was observed but no reactivity with other strains in the panel could be discerned (Fig 4D).


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)

Reactivity of anti-rHpuA and anti-rHpuB antisera with HpuAB in meningococcal lysates.Meningococcal cells of strain 8047, an isogenic Δhpu mutant and two phase variants of a carriage strain (N88.1, hpu-OFF; and N272.1, hpu-ON) were grown to mid-log (OD600 = ~0.5) before 30 μM of desferal was added to produce iron-limited conditions. Cultures were also grown concurrently in iron-replete conditions. All cultures were incubated for two hours before heat-inactivation at 56°C overnight. Lysates were prepared using an equal number of OD units and subject to SDS-PAGE electrophoresis and Western blotting. Blots were then probed with 1:500 dilutions of mouse polyclonal sera followed by a 1:2000 dilution of an anti-mouse IgG HRP-conjugate:- upper panel, anti-rN88-HpuA; middle panel, anti-r8047-HpuA; lower panel anti-r8047-HpuB. Lysates were from:- strain N88.1, induced (lane 1); 8047ΔhpuAB, induced (lane 2); wild-type 8047, uninduced (lane 3); wild-type 8047, induced (lane 4); N272.1 uninduced (lane 5); and N272.1 induced (lane 6).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133855.g003: Reactivity of anti-rHpuA and anti-rHpuB antisera with HpuAB in meningococcal lysates.Meningococcal cells of strain 8047, an isogenic Δhpu mutant and two phase variants of a carriage strain (N88.1, hpu-OFF; and N272.1, hpu-ON) were grown to mid-log (OD600 = ~0.5) before 30 μM of desferal was added to produce iron-limited conditions. Cultures were also grown concurrently in iron-replete conditions. All cultures were incubated for two hours before heat-inactivation at 56°C overnight. Lysates were prepared using an equal number of OD units and subject to SDS-PAGE electrophoresis and Western blotting. Blots were then probed with 1:500 dilutions of mouse polyclonal sera followed by a 1:2000 dilution of an anti-mouse IgG HRP-conjugate:- upper panel, anti-rN88-HpuA; middle panel, anti-r8047-HpuA; lower panel anti-r8047-HpuB. Lysates were from:- strain N88.1, induced (lane 1); 8047ΔhpuAB, induced (lane 2); wild-type 8047, uninduced (lane 3); wild-type 8047, induced (lane 4); N272.1 uninduced (lane 5); and N272.1 induced (lane 6).
Mentions: Both anti-rHpuA mouse antisera bound to a protein of the expected size (~40-kDa) in Western blots of meningococcal lysates of strain 8047 and CC174 hpuAB-ON variant (N272.1) but not the 8047ΔhpuAB mutant or the CC174 hpuAB-OFF variant (N88.1), indicating specific reactivity with HpuA (Fig 3). Surface expression of HpuA in the 8047 and N272.1 strains was demonstrated by detection of high reactivity to formalin-fixed cells in the FACs assays (Fig 4A and 4B). No difference was observed, by Western blotting or FACs, in binding of anti-r8047-HpuA antibodies to strains N272.1 and 8047 whereas the anti-rN88-HpuA sera exhibited a major reduction in reactivity with 8047-HpuA as compared to N88.1-HpuA. This suggests that there are either differences in the level of HpuA expression, with a lower level of expression in strain 8047 as compared to the CC174 strain, or differences in the amounts of variant specific antibodies in each serum (i.e. more variant-specific antibodies in the N88 versus the 8047 sera). Further testing of the reactivity of anti-HpuA antisera to a panel of carriage strains in colony immunoblots showed specific reactivity of anti-rN88-HpuA to only the cognate CC-174 strains, N46 and N52 (Fig 4E). These CC-174 isolates were isolated from the same hall of residence and represent a cluster of genotypically identical strains that are thought to be a product of clonal expansion within the hall of residence [2]. A very weak interaction between anti-r8047-HpuA antibodies and the CC-174 strains was observed but no reactivity with other strains in the panel could be discerned (Fig 4D).

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