Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis.
Lactoferrin-binding activity in Neisseria meningitidis was detected by a solid-phase binding assay with horseradish peroxidase-conjugated human lactoferrin (HRP-lactoferrin). Expression of lactoferrin-binding activity was regulated by the level of iron in the medium, so that growth in the presence of the iron chelator EDDA (ethylenediamine di-ortho-hydroxyphenylacetic acid) resulted in a greater than 350-fold increase in binding activity, which was reversed by addition of excess iron. A maximal level of expression could be obtained at reasonable culture densities by using either intermediate levels of EDDA or high levels of EDDA and moderate levels of complexed iron sources such as hemoglobin and transferrin. Competition binding assays demonstrated that the binding of lactoferrin was specific for human lactoferrin in that neither bovine lactoferrin, human transferrin, nor human hemoglobin was able to block binding of HRP-lactoferrin. The binding specificity for human lactoferrin correlated with growth studies in which human but not bovine lactoferrin could support the growth of iron-starved cells. Binding of lactoferrin was not dependent on its level of iron saturation, since iron-saturated lactoferrin and apolactoferrin were equally effective at blocking binding of HRP-lactoferrin in competitive binding assays. The lactoferrin-binding protein was identified as a 105,000-molecular-weight iron-regulated outer membrane protein in three different meningococcal strains by a batch affinity method with biotinylated human lactoferrin and streptavidin-agarose.
D001059:Apoproteins / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008970:Molecular Weight; D009345:Neisseria meningitidis / Q000378:metabolism*
D001059:Apoproteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; C027768:lactoferrin binding proteins, bacterial; D007501:Iron