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Isolation and characterization of the haemin-binding proteins from Neisseria meningitidis
1Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada.
Abstract:
The mechanism of haem-iron acquisition in Neisseria meningitidis is poorly understood. Using haemin-agarose in a batch affinity chromatography method, two haemin-binding proteins of 97 and 50 kDa were isolated from total membranes derived from Neisseria meningitidis B16B6 grown under iron-deficient but not under iron-replete conditions. No binding proteins were affinity-purified when total membranes underwent limited proteolysis with trypsin, suggesting a haem-protein interaction. When biotinylated human haemoglobin was used as the affinity ligand, proteins of identical molecular mass were isolated. Detection of haemin-binding proteins in a whole cell binding assay demonstrated a surface-exposed location. Competitive binding studies indicated that this haem-protein interaction was specific, because only haemin or human haemoglobin, but not cytochrome c111, protoporphyrin IX, iron-loaded human lactoferrin, iron-loaded human transferrin or Fe(NO3)3, could abrogate binding. The presence of similar haemin-binding proteins in a limited survey of clinical meningococcal strains indicated that the expression of the haemin-binding proteins is not serogroup-specific.
Insights
Neisseria meningitidis acquires iron through specific surface proteins that bind haemin and human haemoglobin. These proteins are expressed under iron deficiency and are present in clinical strains, suggesting a conserved mechanism for iron uptake.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The mechanism of iron acquisition in Neisseria meningitidis is not well understood.
- Iron is essential for bacterial growth and virulence.
Purpose of the Study:
- To identify and characterize proteins involved in haemin-iron acquisition in Neisseria meningitidis.
- To determine the location and specificity of these haemin-binding proteins.
Main Methods:
- Batch affinity chromatography using haemin-agarose to isolate binding proteins from bacterial membranes.
- Limited proteolysis with trypsin to assess the nature of the protein-haemin interaction.
- Affinity purification using biotinylated human haemoglobin as a ligand.
- Whole cell binding assays to determine protein localization.
- Competitive binding studies to assess the specificity of the interaction.
Main Results:
- Two haemin-binding proteins (97 and 50 kDa) were isolated from iron-deficient Neisseria meningitidis membranes.
- Proteolysis experiments suggested a haem-protein interaction.
- Identical proteins were isolated using human haemoglobin as a ligand.
- Binding proteins were surface-exposed.
- Binding was specific for haemin and human haemoglobin, but not other porphyrins or iron-binding proteins.
- Similar proteins were found in clinical meningococcal strains, independent of serogroup.
Conclusions:
- Neisseria meningitidis possesses specific, surface-exposed proteins that bind haemin and human haemoglobin for iron acquisition.
- This mechanism appears conserved across different clinical strains.
- The identified proteins are potential targets for understanding and combating meningococcal infections.