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.     

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.     

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 Negatively Charged Regions of Lactoferrin Binding Protein B,an Adaptation against Anti-Microbial Peptides

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.     

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.     

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.     

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.     

Lactoferrin receptors in Gram-negative bacteria: Insights into the iron acquisition process

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.     

Perspectives on interactions between lactoferrin and bacteria.

Lactoferrin has long been recognized for its antimicrobial properties, initially attributed primarily to iron sequestration. It has since become apparent that interaction between the host and bacteria is modulated by a complex series of interactions between lactoferrin and bacteria, lactoferrin and bacterial products, and lactoferrin and host cells. The primary focus of this review is the interaction between lactoferrin and bacteria, but interactions with the lactoferrin-derived cationic peptide lactoferricin will also be discussed. We will summarize what is currently known about the interaction between lactoferrin (or lactoferricin) and surface or secreted bacterial components, comment on the potential physiological relevance of the findings, and identify key questions that remain unanswered.     

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.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Contribution of bovine lactoferrin inter-lobe region to iron binding stability and antimicrobial activity against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The investigation of the recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against Staphylococcus aureus. rBLfA containing N-lobe (amino acid residues 1–333) and inter-lobe region (residues 334–344) was expressed in Pichia pastoris at shaking flask and fermentor level. The recombinant intact bovine lactoferrin (rBLf) and N-lobe (rBLfN) were expressed in the same system as control. The physical–chemical parameters of rBLfA, rBLfN and rBLf including amino acid residues, molecular weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature than rBLf and rBLfN. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released iron in the pH range of 7.0–3.5, whereas rBLfN lost its iron over the pH range of 7.0–4.0 and iron release from rBLf occurred in the pH range of 5.5–3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5 μmol/L, compared with 12.5 and 25 μmol/L that of rBLfN and rBLf, respectively. These results revealed that S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related positively to the higher anti-S. aureus activity.     

Antimicrobial Functions of Lactoferrin Promote Genetic Conflicts in Ancient Primates and Modern Humans

Lactoferrin is a multifunctional mammalian immunity protein that limits microbial growth through sequestration of nutrient iron. Additionally, lactoferrin possesses cationic protein domains that directly bind and inhibit diverse microbes. The implications for these dual functions on lactoferrin evolution and genetic conflicts with microbes remain unclear. Here we show that lactoferrin has been subject to recurrent episodes of positive selection during primate divergence predominately at antimicrobial peptide surfaces consistent with long-term antagonism by bacteria. An abundant lactoferrin polymorphism in human populations and Neanderthals also exhibits signatures of positive selection across primates, linking ancient host-microbe conflicts to modern human genetic variation. Rapidly evolving sites in lactoferrin further correspond to molecular interfaces with opportunistic bacterial pathogens causing meningitis, pneumonia, and sepsis. Because microbes actively target lactoferrin to acquire iron, we propose that the emergence of antimicrobial activity provided a pivotal mechanism of adaptation sparking evolutionary conflicts via acquisition of new protein functions.     

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.     

Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin

Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.     

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.     

乳铁蛋白的多功能生物活性与临床进展

目前,为应对抗生素耐药性问题,需要探索更多的抗生素替代物用于对抗微生物的感染。乳铁蛋白是一类源自哺乳动物乳汁或其他粘膜分泌的一种糖基化蛋白,具有广谱抗微生物活性、免疫调节以及抗癌作用。乳铁蛋白对于易受感染的早产儿也有很好的耐受作用,基本无不良反应,具有良好的开发前景。本文主要概述乳铁蛋白的基本性质、多功能的生物学活性和作用机制,并探讨牛乳铁蛋白与talactoferrin的临床研究情况。     

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.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.     

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.