Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.     

High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin

The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0x10(18) M(-1). These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane.     

Preparation and Characterization of Neisseria meningitidis Mutants Deficient in Production of the Human Lactoferrin-Binding Proteins LbpA and LbpB

Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285–297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.     

Opposing selective forces for expression of the gonococcal lactoferrin receptor

All isolates of Neisseria gonorrhoeae express receptors that bind human transferrin (Tf). Although lactoferrin (Lf) is abundant on mucosa and in purulent exudates, many gonococci do not express an Lf receptor. The naturally occurring Lf receptor deletion mutant FA1090 (LbpB-LbpA-) is infectious, but a Tf receptor mutant of FA1090 is unable to infect male volunteers [Cornelissen, C.N., Kelley, M., Hobbs, M.M., Anderson, J.E., Cannon, J.G., Cohen, M.S., and Sparling, P.F. (1998) Mol Microbiol 27: 611-616]. Here, we report that expression of an Lf receptor in the absence of the Tf receptor was sufficient for infection, and that expression of both Lf and Tf receptors resulted in a competitive advantage over a strain that made only the Tf receptor in mixed infection of male volunteers. We confirmed that nearly 50% of clinical isolates do not make an Lf receptor. Surprisingly, about half of geographically diverse Lf - isolates representing many different auxotypes and porin serovars carried an identical lbpB lbpA deletion. Among Lf+ strains, all produced the integral outer membrane protein LbpA, but 70% did not express the lipoprotein LbpB. Thus, there are apparently selective pressures for expression of the Lf receptor in the male urethra that are balanced by others against expression of the Lf receptor in niches other than the male urethra.     

The role of lactoferrin binding protein B in mediating protection against human lactoferricin

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.     

Patterns of sequence variation within the Neisseria meningitidis lactoferrin binding proteins

Lactoferrin binding proteins A and B (LbpA and LbpB) compose the lactoferrin receptor of the obligate human pathogen Neisseria meningitidis . This receptor is thought to be important for colonization and initiation of invasive disease because of its role in acquiring host iron and providing protection from the cationic peptide, lactoferricin. By virtue of its function, the receptor is accessible to the host immune system and displays substantial sequence variation. In this study, we analyzed a broad collection of LbpAs (62) and LbpBs (101) to determine the distribution of sequence variation within each protein and to search for patterns between sequence similarity and strain typing. The sequence variation in LbpA was predominantly observed in 3 surface loops and, surprisingly, in the N-terminal region immediately upstream of the predicted TonB box. The analysis of LbpB revealed that the variability was distributed throughout the protein, particularly in the highly variable negatively charged regions in the C-lobe, but otherwise was greater in the N-lobe than the C-lobe. There was no readily identifiable correlation between the sequence variation within LbpA, LbpB, multi-locus sequence type, or serogroup.     

The lactoferrin receptor complex in gram negative bacteria

Bacteria that inhabit the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment on the mucosal surface where iron is complexed by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic anti-microbial peptide lactoferricin is produced by proteolysis of lactoferrin. Several members of the Neisseriaceae and Moraxellaceae families express surface receptors, capable of specifically binding host lactoferrin and extracting the iron from lactoferrin as a source of iron for growth. The receptor is comprised of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a largely exposed surface lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for mediating growth using lactoferrin as a sole iron source whereas LbpB only plays a facilitating role. LbpB, with the presence of a large tract of negatively charged residues, appears to protect the bacterial cell from the bactericidal effects of the lactoferricin. The lactoferrin receptors in these species appear to be essential for survival and thus may serve as potential vaccine targets.     

Molecular characterization of LbpB, the second lactoferrin-binding protein of Neisseria meningitidis

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.     

Characterization of a ferric-binding protein mutant in Haemophilus influenzae

Ferric-binding proteins (FbpA) have been implicated in the transferrin receptor-mediated iron acquisition pathways of Haemophilus influenzae and Neisseria spp. These proteins are believed to function by shuttling iron from outer membrane transferrin receptors to a specific inner membrane permease complex. However, the role of these proteins has not been conclusively resolved, as attempts at creating isogenic mutants in the fbpA genes of both species have been unsuccessful, prompting the hypothesis that FbpA may play a critical role in H . influenzae and Neisseria spp. This study describes the construction and characterization of an H . influenzae isogenic fbpA mutant. It is demonstrated that this mutant is deficient in its ability to use human transferrin as a sole iron source, even though the strain is still competent for binding human transferrin. It is also demonstrated that this mutant is impaired in its ability to use ferric citrate as an iron source, and grows at a reduced rate relative to wild type in broth supplemented with protoporphyrin rather than haemin.     

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.     

Structural Characterization of the Lactoferrin Receptor from Neisseria meningitidis

The meningococcal lactoferrin receptor is composed of the integral outer membrane protein LbpA and the peripheral lipoprotein LbpB. Homooligomeric complexes of LbpA and heterooligomers consisting of LbpA and LbpB were identified. Furthermore, five cell surface-exposed loops of LbpA were identified, which partially confirms a previously proposed topology model.     

Presence of ferric hydroxide clusters in mutants of Haemophilus influenzae ferric ion-binding protein A

The periplasmic iron binding protein plays an essential role in the iron uptake pathway of Gram-negative pathogenic bacteria from the Pasteurellaceae and Neisseriaceae families and is critical for survival of these pathogens within the host. In this study, we report the crystal structures of two mutant forms of ferric ion-binding protein A (FbpA) from Haemophilus influenzae with bound multinuclear oxo-metal clusters. Crystals of site-directed mutants in the metal or anion binding ligands contain protein in the open conformation, and two mutant FbpAs, H9A and N175L, contain different cluster arrangements in the iron-binding pocket. The iron clusters are anchored by binding to the two tyrosine ligands (Tyr195 and Tyr196) positioned at the vertex of the iron-binding pocket but are not coordinated by the other metal binding ligands. Our results suggest that the metal clusters may have formed in situ, suggesting that the mutant FbpAs may serve as a simple model for protein-mediated mineralization.     

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.     

Response of Neisseria meningitidis to iron limitation

In the human body, the concentration of free iron is limiting for bacterial growth, since iron is bound to transport and storage proteins such as transferrin and lactoferrin. When grown under iron starvation, Neisseria meningitidis produces receptors for these proteins in the outer membrane. These receptors are presently being characterized at the molecular level. Here, we summarize our current knowledge of these receptors, with special emphasis on the LbpA and FrpB proteins, which are studied in our laboratories. Furthermore, the genetic and antigenic variability of these proteins and their vaccine potential are discussed.     

"Dilysine trigger" in transferrins probed by mutagenesis of lactoferrin: crystal structures of the R210G,R210E,and R210L mutants of human lactoferrin

The mammalian iron-binding proteins lactoferrin (Lf) and transferrin (Tf) bind iron very tightly, but reversibly. Despite homologous structures and essentially identical iron binding sites, Tf begins to release iron at pH 6.0, whereas Lf retains iron to pH approximately 3.5. This difference in iron retention gives the two proteins different biological roles. Two lysine residues, Lys 206 and Lys 296, which form a hydrogen-bonded dilysine pair in human Tf, have been shown to strongly influence iron release from the N-lobe. The equivalent residues in human Lf are Arg 210 and Lys 301, and we have here mutated Arg 210 in the N-lobe half-molecule of human lactoferrin, Lf(N), to probe its role in iron release. The Lf(N) mutants R210G, R210E, and R210L were expressed, purified, and crystallized, and their crystal structures were determined and refined at resolutions of 1.95 A (R210G), 2.2 A (R210E), and 2.0 A (R210L). The overall structures are very similar to that of wild-type Lf(N), but with small differences in domain orientations. In each of the mutants, however, Lys 301 (equivalent to Lys 296 in Tf) changes its conformation to fill the space occupied by Arg 210 Neta2 in wild-type Lf(N), interacting with the two tyrosine ligands Tyr 92 and Tyr 192. By comparison with other Lf and Tf structures, we conclude that Lys 301 (or Lys 296 in Tf) only occupies this site when residue 210 (206 in Tf) is nonpositive (neutral as in R210G and R210L or negative as in R210E). Thus, Lys 206 in the Tf dilysine pair is identified as having a depressed pK(a). Three specific sites are variably occupied by polar groups in the Lf mutants and other Lf and Tf proteins, and when coupled with iron-release data, these give new insights into the factors that most influence iron retention at low pH.     

The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.     

Sequence variability of the meningococcal lactoferrin-binding protein LbpB

The lactoferrin receptor of Neisseria meningitidis consists of two proteins, LbpA and LbpB. LbpB is considered a promising vaccine candidate, and therefore its sequence variability was studied. LbpB from five different strains exhibited 70-80% mutual identity at the amino acid level. Most sequence variability was found in two stretches with a high content of negatively charged amino acids. These stretches were sequenced from six additional strains. One of the stretches is of variable length and is missing in some of the strains. The other stretch is present in all strains, but varies considerably in its exact amino acid sequence. The high degree of variability is disadvantageous for vaccine development, but may be useful for epidemiological studies.     

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.     

Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.