Limits...
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

Utilization of Hb and Tf by wild-type and mutant N. meningitidis strains.Desferal (40 μg/ml) was added to molten MH agar to chelate available iron in the agar. A suspension of 108 cells from an overnight culture was spread onto each plate before sterile filter discs infused with either 100 μg of Hb or 500 μg of Tf were placed on the agar. Plates were incubated at 37°C, 5% CO2 for 24 hours. Discs infused with FeCl3 and PBS were used as positive and negative controls. Panel (A) MC58; panel (B) 8047.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4514712&req=5

pone.0133855.g001: Utilization of Hb and Tf by wild-type and mutant N. meningitidis strains.Desferal (40 μg/ml) was added to molten MH agar to chelate available iron in the agar. A suspension of 108 cells from an overnight culture was spread onto each plate before sterile filter discs infused with either 100 μg of Hb or 500 μg of Tf were placed on the agar. Plates were incubated at 37°C, 5% CO2 for 24 hours. Discs infused with FeCl3 and PBS were used as positive and negative controls. Panel (A) MC58; panel (B) 8047.

Mentions: In order to investigate the contributions of the haemoglobin binding proteins of N. meningitidis to the ability of meningococci to cause invasive disease, parts of the hmbR and hpuAB loci were deleted and replaced by antibiotic cassettes. Mutations were constructed in a strain with only the HmbR receptor, strain MC58, and one, strain 8047, with both receptors (i.e. HmbR and HpuAB). The tbpBA genes were also inactivated by a similar method in both wild-type (wt) and hmbR mutant backgrounds. The repeat tracts of the Hb receptors in the wt strains and tbpBA mutants were sequenced, HmbR was in the ON state in the MC58 strain-background but OFF in strain 8047 wherein HpuAB was in an ON state. Growth assays showed similar doubling times for the wt and mutant strains in MH broth (data not shown). Phenotypic characterisation of wt and mutant MC58 strains was then performed using a disc diffusion assay. The MC58ΔhmbR utilized Tf but not Hb whilst the converse was observed for MC58ΔtbpBA (Fig 1A). The MC58 double mutant was unable to utilize Tf or Hb whilst the wt strain utilised both (Fig 1A). Similarly, only the 8047ΔhmbRΔhpuAB mutant was unable to utilise Hb while all 8047ΔtbpBA mutants failed to grow when Tf was the sole iron source (Fig 1B). The ability of ΔhpuAB mutants to utilise Hb could be due to the presence of small numbers of hmbR-ON phase variants in the inoculum. All strains utilized free iron provided in the form of 0.1 M FeCl3 (Fig 1). The hemO gene is located upstream of the hmbR gene and HemO is required for utilisation of haem derived from Hb [45]. All mutant strains are assumed to have retained HemO function as indicated by the ability of 8047ΔhmbR to grow on Hb presumably through the combined actions of HpuAB and HemO.


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)

Utilization of Hb and Tf by wild-type and mutant N. meningitidis strains.Desferal (40 μg/ml) was added to molten MH agar to chelate available iron in the agar. A suspension of 108 cells from an overnight culture was spread onto each plate before sterile filter discs infused with either 100 μg of Hb or 500 μg of Tf were placed on the agar. Plates were incubated at 37°C, 5% CO2 for 24 hours. Discs infused with FeCl3 and PBS were used as positive and negative controls. Panel (A) MC58; panel (B) 8047.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133855.g001: Utilization of Hb and Tf by wild-type and mutant N. meningitidis strains.Desferal (40 μg/ml) was added to molten MH agar to chelate available iron in the agar. A suspension of 108 cells from an overnight culture was spread onto each plate before sterile filter discs infused with either 100 μg of Hb or 500 μg of Tf were placed on the agar. Plates were incubated at 37°C, 5% CO2 for 24 hours. Discs infused with FeCl3 and PBS were used as positive and negative controls. Panel (A) MC58; panel (B) 8047.
Mentions: In order to investigate the contributions of the haemoglobin binding proteins of N. meningitidis to the ability of meningococci to cause invasive disease, parts of the hmbR and hpuAB loci were deleted and replaced by antibiotic cassettes. Mutations were constructed in a strain with only the HmbR receptor, strain MC58, and one, strain 8047, with both receptors (i.e. HmbR and HpuAB). The tbpBA genes were also inactivated by a similar method in both wild-type (wt) and hmbR mutant backgrounds. The repeat tracts of the Hb receptors in the wt strains and tbpBA mutants were sequenced, HmbR was in the ON state in the MC58 strain-background but OFF in strain 8047 wherein HpuAB was in an ON state. Growth assays showed similar doubling times for the wt and mutant strains in MH broth (data not shown). Phenotypic characterisation of wt and mutant MC58 strains was then performed using a disc diffusion assay. The MC58ΔhmbR utilized Tf but not Hb whilst the converse was observed for MC58ΔtbpBA (Fig 1A). The MC58 double mutant was unable to utilize Tf or Hb whilst the wt strain utilised both (Fig 1A). Similarly, only the 8047ΔhmbRΔhpuAB mutant was unable to utilise Hb while all 8047ΔtbpBA mutants failed to grow when Tf was the sole iron source (Fig 1B). The ability of ΔhpuAB mutants to utilise Hb could be due to the presence of small numbers of hmbR-ON phase variants in the inoculum. All strains utilized free iron provided in the form of 0.1 M FeCl3 (Fig 1). The hemO gene is located upstream of the hmbR gene and HemO is required for utilisation of haem derived from Hb [45]. All mutant strains are assumed to have retained HemO function as indicated by the ability of 8047ΔhmbR to grow on Hb presumably through the combined actions of HpuAB and HemO.

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