Related Experiment Videos
Molecular characterization of LbpB, the second lactoferrin-binding protein of Neisseria meningitidis
A Pettersson1, T Prinz, A Umar
1Department of Molecular Cell Biology and Institute of Biomembranes, Utrecht University, The Netherlands. A.M.Pettersson-Fernholm@biol.ruu.nl
Abstract:
The lbpA gene of Neisseria meningitidis encodes an outer membrane lactoferrin-binding protein and shows homology to the transferrin-binding protein, TbpA. Previously, we have detected part of an open reading frame upstream of lbpA. The putative product of this open reading frame, tentatively designated lbpB, showed homology to the transferrin-binding protein TbpB, suggesting that the lactoferrrin receptor, like the transferrin receptor, consists of two proteins. The complete nucleotide sequence of lbpB was determined. The gene encodes a 77.5 kDa protein, probably a lipoprotein, with homology, 33% identity to the TbpB of N. meningitidis. A unique feature of LbpB is the presence of two stretches of negatively charged residues, which might be involved in lactoferrin binding. Antisera were raised against synthetic peptides corresponding to the C-terminal part of the putative protein and used to demonstrate that the gene is indeed expressed. Consistent with the presence of a putative Fur binding site upstream of the lbpB gene, expression of both LbpA and LbpB was proved to be iron regulated in Western blot experiments. The LbpB protein appeared to be less stable than TbpB in SDS-containing sample buffer. Isogenic mutants lacking either LbpA or LbpB exhibited a reduced ability to bind lactoferrin. In contrast to the lbpB mutant, the lbpA mutant was completely unable to use lactoferrin as a sole source of iron.
Insights
Neisseria meningitidis uses two outer membrane proteins, LbpA and LbpB, to bind lactoferrin. Both proteins are essential for iron acquisition, with LbpA being critical for lactoferrin utilization.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Neisseria meningitidis utilizes outer membrane proteins for nutrient acquisition.
- The lactoferrin receptor system in N. meningitidis was previously incompletely characterized.
- Homology suggests a two-protein system for lactoferrin binding, similar to the transferrin receptor.
Purpose of the Study:
- To fully characterize the lbpB gene and its protein product.
- To investigate the roles of LbpA and LbpB in lactoferrin binding and iron uptake.
- To determine the regulation of the lactoferrin receptor system.
Main Methods:
- Nucleotide sequencing of the lbpB gene.
- Protein expression analysis using antisera and Western blotting.
- Construction and analysis of isogenic mutants (lbpA and lbpB knockouts).
- Iron-binding and utilization assays.
Main Results:
- The lbpB gene encodes a 77.5 kDa lipoprotein (LbpB) homologous to TbpB.
- LbpB possesses unique negatively charged regions potentially involved in lactoferrin binding.
- Expression of both LbpA and LbpB is iron-regulated, with a putative Fur binding site upstream of lbpB.
- Mutants lacking LbpA or LbpB showed reduced lactoferrin binding; the lbpA mutant was unable to use lactoferrin for iron.
Conclusions:
- The lactoferrin receptor in N. meningitidis comprises two proteins, LbpA and LbpB.
- Both LbpA and LbpB are crucial for lactoferrin binding and iron acquisition.
- LbpA is essential for utilizing lactoferrin as an iron source, while LbpB contributes to binding.