Lactoferrin receptors in gram-negative bacteria: insights into the iron acquisition process

• Published in: Biometals 2004-06, Vol.17 (3), p.235-243
• Main Authors: Ekins, Andrew, Khan, Ali G, Shouldice, Stephen R, Schryvers, Anthony B
• Format: Article
• Language: English
• Subjects: Amino acids; Anti-Infective Agents - metabolism; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystallography; Haemophilus influenzae; Haemophilus influenzae - chemistry; Haemophilus influenzae - genetics; Haemophilus influenzae - metabolism; Humans; Iron; Iron - metabolism; Membranes; Models, Molecular; Mutagenesis, Site-Directed; Mutants; Neisseria meningitidis; Neisseria meningitidis - genetics; Neisseria meningitidis - metabolism; Pathogens; Protein Structure, Tertiary; Proteins; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Transferrin - metabolism; Transport processes
• ISSN: 0966-0844; 1572-8773
• DOI: 10.1023/B:BIOM.0000027698.43322.60

One component of the anti-microbial function of lactoferrin (Lf) is its ability to sequester iron from potential pathogens. To overcome this iron limitation, a number of gram-negative bacterial pathogens have developed a mechanism for acquiring iron directly from this host glycoprotein. This mechanism involves surface receptors capable of specifically binding Lf from the host, removing iron and transporting it across the outer membrane. The iron is then bound by a periplasmic iron-binding protein, FbpA, and transported into the cell via an inner membrane complex comprised of FbpB and FbpC. The receptor has been shown to consist of two proteins, LbpA and LbpB. LbpB is bilobed lipoprotein anchored to the outer membrane via fatty acyl groups attached to the N-terminal cysteine. LbpA is a homologue of siderophore receptors, which consist of an N-terminal plug and a C-terminal beta-barrel region. We propose that the receptor proteins, LbpA and LbpB, induce conformational changes in human Lf (hLf) that lower its affinity for iron that binding by FbpA can drive the transport across the outer membrane, a mechanism shared with transferrin (Tf) receptors. The interaction between the receptor proteins and Lf is quite extensive and has been previously studied by using chimeric proteins comprised of Lf & Tf. In an attempt to evaluate the role of FbpA in the transport process, a series of site-directed mutants of FbpA were prepared and used to replace the wild-type protein in the iron acquisition pathway. The mutations were made in the iron-binding and anion-binding ligands of FbpA and were designed to result in altered binding properties. Protein crystallography of the iron-bound form of the Q58L mutant protein revealed that it was in the open conformation with iron coordinated by Y195 and Y196 from the C-terminal domain but not by the other iron-liganding amino acids from the N-terminal domain, H9 and E57. Replacement of the native FbpA in Neisseria meningitidis with wild-type or mutant Haemophilus influenzae FbpAs resulted in a defect in growth on Tf or Lf, suggesting that there may be a barrier to functional expression of H. influenzae FbpAs in Neisseria meningitidis. Thus mutants of the N. meningitidis FbpA are being prepared to replace wild-type protein in order to test their ability to mediate transport from hLf.