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Future Microbiology
Review
Bacterial receptors for host transferrin and lactoferrin: molecular mechanisms and role in host–microbe interactions Ari Morgenthau1, Anastassia Pogoutse2, Paul Adamiak1, Trevor F Moraes2 & Anthony B Schryvers*1,3 Department of Microbiology, Immunology & Infectious Diseases, Health Sciences Centre, 3330 Hospital Drive Northwest Calgary, Alberta, T2N 4N1, Canada 2 Department of Biochemistry, Medical Sciences Building, King’s College Circle, Toronto, Ontario, M5S 1A8, Canada 3 Department of Biochemistry & Molecular Biology, Health Sciences Centre, 3330 Hospital Drive Northwest Calgary, Alberta, T2N 4N1, Canada *Author for correspondence: Tel.: +1 403 220 3703 n Fax: +1 403 210 9747 n [email protected] 1
Iron homeostasis in the mammalian host limits the availability of iron to invading pathogens and is thought to restrict iron availability for microbes inhabiting mucosal surfaces. The presence of surface receptors for the host iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) in globally important Gramnegative bacterial pathogens of humans and food production animals suggests that Tf and Lf are important sources of iron in the upper respiratory or genitourinary tracts, where they exclusively reside. Lf receptors have the additional function of protecting against host cationic antimicrobial peptides, suggesting that the bacteria expressing these receptors reside in a niche where exposure is likely. In this review we compare Tf and Lf receptors with respect to their structural and functional features, their role in colonization and infection, and their distribution among pathogenic and commensal bacteria. Role of bacterial receptors for transferrin & lactoferrin in colonization & infection
Due to the critical role that iron plays in mammalian metabolism and its potential toxicity, mammals have developed effective systems for handling iron and regulating levels of available iron [1]. The host iron-binding protein transferrin (Tf) plays a central role in systemic iron homeostasis by shuttling iron beween cells that supply iron to those that require it. Dietary iron acquired from gut enterocytes or recycled erythrocyte iron processed by macrophages or hepatocytes is loaded onto Tf through the combined action of the ferrous iron transporter ferroportin, which exports iron from the cell, and a ferroxidase (hephestin or ceruloplasmin), which converts ferrous iron to ferric ion in order to be bound by Tf. These processes are regulated by hepcidin, a key regulatory molecule in iron homeostasis, which influences the stability of ferroportin [2]. The iron-loaded (holo)-Tf supplies iron to various cells throughout the body via a process initiated by the binding of holo‑Tf to the human Tf (hTf ) receptor TFR1. The bound holo‑Tf is internalized by receptor-mediated endocytosis, and iron removal is facilitated by the acidic environment of the endosome. The receptor-bound iron-free apo-Tf is returned to 10.2217/FMB.13.125 © 2013 Future Medicine Ltd
the surface by fusion of the receptor-containing vesicle with the cytoplasmic membrane, where the apo‑Tf dissociates from the TFR1 receptor at the physiological pH of the extracellular milieu. Tf can also be endocytosed upon binding to TFR2 (HFE3) in some tissue types, but iron delivery is not thought to be the primary function of this receptor. In normal plasma, Tf is only approximately 30% iron-saturated and serves to readily sequester any nonheme iron that is released into the extracellular environment within the body [3]. The iron sequestration by Tf reduces the levels of free iron below that required for bacterial growth [4]. The structurally related iron-binding glycoprotein lactoferrin (Lf) serves a similar role, but is thought to primarily act on mucosal surfaces and sites of inflammation [5]. Lf has a higher affinity for iron than Tf, particularly at the lower pH at sites of inflammation. Lf is secreted in its apo form by epithelial cells (Figure 1) into most exocrine secretions and is released from secretory granules of neutrophils at sites of inflammation. Proteolytic cleavage of Lf leads to the release of a cationic antimicrobial peptide, lactoferricin, derived from the N-terminus. Lf is present at very low levels in tissue interstitial fluids, but can be found at high levels at sites of inflammation. Future Microbiol. (2013) 8(12), 1575–1585
Keywords bacterial receptors n iron acquisition n iron homeostasis n lactoferrin n transferrin n
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Figure 1. Availability of transferrin and lactoferrin on mucosal surfaces. Lf is released from secondary granules of neutrophils at sites of inflammation and can provide Lf on the mucosal surface due to neutrophil migration. Lf is also produced directly by epithelial glandular cells for secretion onto mucosal surfaces. Tf in serum could be released onto mucosal surfaces by episodic bleeding, but the route for delivering Tf to the mucosal surface from the submucosal space has not been determined. Tf in the submucosal space can be taken in by receptor-mediated endocytosis to provide iron to the epithelial cells, and some bacteria have been shown to hijack the recycling pathway to deliver Tf to the epithelial surface (yellow-colored bacterium). Lf: Lactoferrin; Tf: Transferrin.
Similarly, Lf is normally found at low levels in serum (0.4–2 mg/l), but can increase up to 200 mg/l during sepsis [6]. Owing to this, there is a generally held view that Tf is the major iron-binding protein within the body and Lf is the dominant iron-binding protein on mucosal surfaces. Gram-negative bacteria from the families Neisseriaceae, Moraxellaceae and Pasteurellaceae primarily reside in the upper respiratory or genitourinary tract. In humans, they constitute major causes of meningitis (Haemophilus influen zae and Neisseria meningitidis), upper and lower respiratory infections (Moraxella catarrhalis and H. influenzae) and genitourinary infections (Neisseria gonorrhoeae) [7–9]. In cattle, they are the primary cause of the bovine respiratory disease complex (Mannheimia haemolytica, Pasteurella multocida and Histophilus somni) plaguing cattle feedlots [10]. Members of Pasteurellaceae (Actinobacillus pleuropneumoniae, Haemophilus parasuis, Actinobacillus suis and P. multocida) predominantly affect the pig industry [11]. A common adaptation that these pathogens share for survival in the respiratory or genitourinary tract of their host is the possession of surface 1576
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receptors that directly bind Tf or Lf to acquire iron for growth [12]. The receptors are exquisitely specific for Tf and Lf from their mammalian host [13], corresponding to the restricted host range of these pathogens. Although the Tf receptors would clearly be advantageous for survival during invasive infection, their ubiquitous presence argues for the availability of Tf on the mucosal surface where the bacteria possessing them primarily reside. However, the mechanisms for the transport of Tf to the mucosal surface have not been established (Figure 1). Gonococcal infection studies in male volunteers demonstrated that survival of the bacteria was dependent upon the presence of either a Tf or Lf receptor, and led to the observation that the mucosal levels of Tf (inferred by measurements of urinary levels) exceed that of Lf prior to challenge [14]. Similarly, the requirement of both protein components of the bacterial Tf receptor for survival of A. pleuro pneumoniae in an aerosol challenge experiment [15] suggests that Tf is an important iron source in the upper respiratory tract of pigs. In contrast to the GI tract where exogenous sources of iron would readily be available, the available iron on the upper respiratory and genitourinary future science group
Bacterial receptors for host transferrin & lactoferrin
tracts would be primarily derived from the host, which may explain why Tf and Lf receptors have only been observed in bacteria colonizing these surfaces. The challenges in obtaining accurate measure ments of the levels of Tf and Lf on mucosal surfaces have resulted in a relative lack of reliable information regarding Tf and Lf on the mucosal surfaces of the upper respiratory and genito urinary tracts. The Tf and Lf receptor-dependent growth of bacteria provides indirect evidence for the availability of these proteins and, at present, is perhaps one of our best sources of information regarding whether there are adequate levels of these proteins to support microbial growth. The mucosal surface is a critical interface between the host and the resident microbiome [16] and there is an increasing recognition of the complex interplay between the host and microbial community. Recently, it has been demonstrated that Helicobacter pylori is able to modulate the Tf recycling pathway in host epithelial cells with CagA and VacA, resulting in transport of submucosal Tf to the epithelial surface [17]. Thus, it will be important to consider the impact of the resident microbiome when evaluating the availability of Tf and Lf on various mucosal surfaces (F igure 1). An additional consideration is that some bacteria are capable of attaching to host surface proteins to mediate transcytosis across epithelial cells, thus they may be able to reside in the submucosal space [18]. The pathogenic Neis seria spp. possess phase-variable Opa adhesion proteins that mediate this process through interaction with CEACAM1 receptors expressed on
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epithelial cells. Tf and Lf receptors and opacityassociated proteins are also found in some of the ‘nonpathogenic’ Neisseria spp. [19], which raises interesting questions regarding whether accessing a unique ecological niche (the submucosal space) and utilization of Tf and Lf could be viewed as a first step towards developing pathogenicity. In other words, some commensal species have attributes shared with the pathogenic species and may be capable of causing disease if additional attributes are acquired. The recent demonstration that one of the Lf receptor proteins, LbpB, protects against the cationic antimicrobial peptide lactoferricin [20] indicates that enhanced survival of bacteria that possess Lf receptors may be due to more than one factor (Box 1). Structural & functional features of Tf & Lf receptors
The canonical Tf receptor in Gram-negative bacteria consists of two proteins that are each capable of binding Tf independently: TbpA and TbpB (Figure 2). TbpB has a strong preference for binding the iron-loaded (holo) form of Tf, whereas TbpA binds both apo- and holo‑Tf with equal affinity. As TbpB is a surfaceexposed lipoprotein, the selective capture of holo‑Tf has been proposed to serve the role of delivering holo‑Tf to the integral outer membrane protein TbpA (Figure 2). With the notable exception of N. meningitidis strain B16B6 [21] or in N. gonorrhoeae TbpA mutants [22], growth on Tf as an iron source under laboratory conditions does not require the presence of TbpB.
Box 1. Availability of transferrin and lactoferrin on mucosal surfaces. Transferrin (Tf) is the predominant iron-containing extracellular protein within the body; therefore, Tf receptors should play a critical role during infection Tf receptors are required for colonization/infection of the male genitourinary tract and the significant levels of measured urinary Tf prior to challenge infers normal availability on the mucosal surface Tf receptors are required for colonization/infection of the porcine upper respiratory tract The mechanism for transport of Tf to the mucosal surface is not known (Figure 1) A mechanism by which bacteria hijack the Tf recycling pathway to bring Tf to the epithelial surface has been described for Helicobacter pylori, raising the spectre of microbial modulation of Tf availability on other mucosal surfaces (Figure 1) Normal physiological levels of lactoferrin (Lf) in serum are low, but due to the inflammatory response during invasive infection, Lf levels may substantially increase, particularly in localized areas Mechanisms for Lf export to the mucosal surface are known (Figure 1) , but the normal levels may be relatively low, as observed in the male genitourinary tract Lf receptors may play an important role during colonization, but the relative importance for iron acquisition and protection from cationic antimicrobial peptides is uncertain Assumptions that Lf receptors were primarily important on mucosal surfaces need to be revisited and the relative role in iron acquisition and protection from cationic antimicrobial peptides needs to be assessed
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Figure 2. Iron uptake mediated by transferrin and lactoferrin receptors. (A) Tf and Lf receptors consist of an integral outer membrane protein (TbpA or LbpA) and a membrane-anchored lipoprotein (TbpB or LbpB). NalP is capable of selectively cleaving the anchor peptide of LbpB. (B) Membrane-anchored TbpB captures iron-loaded Tf with its N-lobe, whereas released LbpB binds to Lf through the negatively charged region in the C-lobe. (C) TbpB delivers Tf to TbpA to initiate the removal and transport of iron to the periplasmic iron-binding protein FbpA. The released LbpB would not facilitate transfer of Lf to LbpA. Lf: Lactoferrin; Tf: Transferrin.
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Thus, the role of TbpB in delivering holo‑Tf to TbpA is thought to only be essential under the more stringent growth conditions encountered in vivo. The first published structure of TbpB from the porcine pathogen A. pleuropneumoniae demonstrated that TbpB is made up of two structurally similar lobes, termed the N-lobe and the C-lobe, which are positioned orthogonally with respect to each other and connected by a flexible linker [23]. Each of the TbpB lobes contains two domains, a b-barrel domain that sits parallel to a handle domain. TbpB also contains an anchor peptide that attaches to the lipid moiety responsible for tethering the protein to the membrane (Figure 2). Only the last (C-terminal) 17 amino acids of the anchor peptide region were resolved in the structure and shown to be intimately associated with the C-terminal lobe, effectively wrapping around the external surface of the C-terminal lobe. The purpose of this unusual association of the anchor peptide with the C-terminal lobe is unknown, but it has been speculated that modulation of the interaction upon binding of holo‑Tf by TbpB could promote interaction of TbpB with TbpA (Figures 2C & 3) [24]. A preliminary model for the complex between TbpB and Tf, involving the TbpB N-lobe and porcine Tf C-lobe, was generated using computational docking and supported by site-directed mutagenesis of the residues from the predicted binding surface of TbpB [23]. Improved models for the interaction of TbpB with Tf were obtained with additional TbpB structures by combining experimental data from hydrogen–deuterium exchange coupled with mass spectrometry experiments and binding assays performed with sitedirected mutants of the TbpBs [25,26]. The development and use of a program that incorporated constraints from the experimental data into the standard Rosetta docking algorithms resulted in models of the complex that involved the same interface, but differed in the orientation of Tf relative to TbpB by 180°. The structure of the complex between N. meningitidis TbpB and hTf determined by protein crystallography confirms this latter orientation and demonstrates that the region of Tfs recognized by TbpBs from diverse strains and species are essentially the same [27]. Interestingly, the binding interface of TbpB on Tf partially overlaps with the interface recognized by the mammalian Tf receptor, indicating that simultaneous binding by both receptors is not possible [3]. The Tf–TbpB structure indicates that the Tf C-lobe is in the closed, iron-loaded conformation, strongly suggesting that TbpB future science group
Review
locks Tf in the iron-loaded form by creating an environment that limits the charge repulsion between two key residues in the C-lobe cleft. The structural results are consistent with the role of TbpB delivering the iron-loaded form of Tf to TbpA at the bacterial cell surface (Figure 2). The recently solved structure of a complex of N. meningitidis TbpA and hTf [28] has provided considerable insights into the iron removal and transport process. TbpA contains a 22-stranded b-barrel encircling a plug domain with Tf bound through the long loops on the extracellular side of the protein. The structure of the TbpA–Tf complex shows the Tf C-lobe to be in a semiopen conformation, with no iron in its binding site. A portion of loop 3 of TbpA, termed the ‘helix finger’, is inserted into the cleft between the C1 and C2 subdomains of the Tf C-lobe, along with a loop extending from the plug domain. Owing to this, it is thought that the conformational change in the Tf C-lobe that occurs upon binding to TbpA lowers the affinity for binding iron, as was originally proposed [29]. Steered molecular dynamics provides a reasonable description of how interaction with TonB in the periplasm could provide a potential path for
TbpB
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TbpB Anchor peptide
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Figure 3. Model of the TbpB–transferrin–TbpA ternary complex. An updated model of the TbpB–transferrin–TbpA complex using structural information from the crystallized TbpB–transferrin complex [20] . In this model, there is a 180° shift in the orientation of TbpB relative to hTf compared with the published model [28] , which positions the junction between the anchoring peptide and N-lobe handle domain, closer to the membrane surface. This orientation provides greater opportunity for interactions between the TbpB-anchoring peptide and TbpA. The arrow signifies a potential interaction between the anchoring peptide of TbpA and TbpB. hTf: Human transferrin. Data taken from [20] .
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transport of iron across the membrane, utilizing the conserved EIEYE motif [28]. However, this does not provide an explanation of what is driving the movement across the membrane, nor how otherwise insoluble ferric iron could be released at the periplasmic face. The possibility that this process is driven by capture of the ferric iron by the apo form of the periplasmic iron binding protein FbpA (Figure 2) through intimate interactions with TbpA is supported by prior stability of unpurified proteins from rates of H/D exchange studies [30] and site-directed mutagenesis studies with FbpA [31]. While the TbpB–Tf and TbpA–Tf structures [27,28] provide insight into how TbpA and TbpB interact with Tf individually, they provide little insight into how TbpA and TbpB work in concert to utilize Tf-bound iron (Box 2). A ternary TbpB–Tf–TbpA complex can be formed from either a TbpA–Tf or a TbpB–Tf complex, but requires the presence of the intact TbpB anchor peptide [24], which is necessary, but not sufficient, for binding. The TbpA and TbpB footprints on Tf are distinct [27,28,32], but TbpB and TbpA bind to the Tf C-lobe in the closed and partially open conformation, respectively, indicating that the TbpB–Tf interaction needs to be altered during the ‘hand-off’ to TbpA. A model for the ternary Tf–TbpA–TbpB complex has been developed by fitting structures of TbpB–Tf and TbpA–Tf complexes into small angle x-ray scattering (SAXS) and negative-stain electron microscopy data obtained from purified ternary complexes [28]. However, the authors did not have the crystallographic data for the N. menin gitidis TbpB–hTf complex at the time, thus they used the structural models of the complex of porcine Tf and A. pleuropneumoniae TbpB derived from a computational docking approach [23] as a template for the Tf–TbpB complex that was fitted into the envelope generated by the SAXS and electron microscopy data. The orientation of
hTf relative to TbpB in the model derived from the SAXS and molecular docking does not agree with the orientation shown in the Tf–TbpB structure obtained via x-ray crystallog raphy [27]. Thus, we have generated a new model of the complex using structural data from the TbpA–Tf and TbpB–Tf complexes (Figure 3). In this model, the alternate orientation of Tf relative to TbpB positions the anchor peptide in a more favorable orientation for interactions with TbpA. Clearly, additional experimental studies need to be directed towards determining the true nature of the ternary complex. In the absence of structural information for intact LbpB, LbpA and complexes with Lf, inferences regarding the structural and mechanistic features are primarily based on comparisons with the structural homologs from the Tf receptor system. It is a reasonable assumption that the LbpA structure will largely resemble TbpA and that the C-lobe of Lf will dock onto LbpA in a similar manner, as has been proposed [33], since prior experiments have established that LbpA binds to both domains of the Lf C-lobe [34]. The high degree of identity in the plug region and the conserved EIEYE motif suggests that the LbpA shares the mechanism for channel formation and iron transport across the membrane with TbpA. By contrast, inferences regarding the interaction between LbpB and Lf, and the role of LbpB in facilitating the iron acquisition process [33] are too speculative at present. To date, efforts to determine the structure of LbpBs have only yielded structures of the LbpB N-lobe [35] and there is no experimental evidence supporting the orthogonal association between the N-lobe and C-lobe that is observed in TbpB. In fact, the lack of structural data on full length LbpB may suggest a degree of flexibility between the N- and C-lobes. The assumption that the Cap region of the N-lobe of LbpB binds to the C-terminal lobe of Lf [33] ignores the obvious tendency for
Box 2. Structural and functional features of transferrin and lactoferrin receptors. The bacterial transferrin (Tf) receptor is composed of the TonB-dependent transporter TbpA and the lipid-anchored protein TbpB TbpB serves to capture holo‑Tf and transfer it to TbpA through the formation of a ternary complex involving the anchor peptide region The details of the process of ternary complex formation and the authentic structure of the ternary complex need to be determined The structure of the Tf–TbpA complex provides insights into the iron removal process and formation of a channel for transport of iron across the membrane, but the role of FbpA in the transport process is uncertain The structure of the TbpB–Tf complex may not be an appropriate model for the LbpB–lactoferrin interaction LbpB is cleaved by NalP, thus it is unlikely to serve a role equivalent to TbpB in iron acquisition
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the positively charged cationic region of the N1 domain of Lf to bind to the negatively charged regions in the C-lobe of LbpB (Figure 2) [20] and that the LbpB–Lf interaction is notably sensitive to pH and salt concentration [36]. This interaction is likely to be responsible for the observed role of LbpB in mediating protection against the cationic antimicrobial peptide lactoferricin [20]. The selective proteolytic release of LbpB from the bacterial surface by NalP [37] in conjunction with evidence for NalP expression in vivo [38,39] indicates that there will likely be excess released LbpB present in the host (Figure 2B & C). Proteo lysis of the anchor peptide makes it unlikely that it will be capable of facilitating the formation of the ternary complex, as has been observed in the mechanism for iron acquisition by the TbpA–TbpB receptor [24]. Thus, released LbpB would not facilitate the iron acquisition process (Figure 2), particularly if free Lf has a comparable affinity for LbpA as the Lbp–Lf complex. These findings all argue against LbpB playing a similar role in the iron acquisition process to TbpB. Thus, it is possible that LbpB has evolved from a protein with a similar role to TbpB into a protein whose primary function is related to protection from cationic peptides, and may have altered its original binding properties in the process. In this context, the observation that over half of the clinical gonococcal isolates are naturally deficient in Lf utilization while there was an observed selective advantage of having a functional lbp operon in the human gonococcal infection model [40] could be revisited from a different perspective. Prevalence & diversity of Tf & Lf receptors
The bacterial Tf receptors were originally identified in important Gram-negative pathogens of humans and food production animals from the Neisseriaceae, Pasteurellaceae and Moraxellaceae families [12]. These pathogens share a similar niche, colonizing the urogenital or respiratory tracts of their respective hosts, which serve as the only reservoirs for these highly host-adapted bacteria. The ubiquitous presence of the Tf receptors in clinical disease isolates from invasive infection argues that these components are important virulence factors by providing a sustained source of iron for growth during infection. However, the selective forces for retention of the Tf receptors are undoubtedly related to survival of the bacteria in the upper respiratory or genitourinary tracts, as was highlighted by the demonstration that the Tf receptor is critical for colonization in a human gonococcal challenge model [14]. The presence of Tf receptors in some commensal pathogenic future science group
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Neisseria spp. supports this hypothesis [41], but the absence of receptors from some commensal strains [19] begs the question of whether the Tf receptor provides a survival advantage primarily in a specialized niche that is only shared by a subset of the commensal bacteria or not. Overall there is greater sequence diversity in the TbpB than TbpA proteins, likely a reflection of the greater surface accessibility of the TbpB protein and a commensurate selective pressure for antigenic variation. Analysis of the distribution of tbpB genes within Neisseria spp. concluded that the phylogenetic clades assorted independently from species [41], suggesting that there may be a common gene pool available to closely related species sharing a particular niche. This is perhaps not surprising considering the common uptake sequences recognized by the natural transformation systems present in these species, although there clearly are various systems that influence horizontal exchange rates [42,43]. This observation also indicates that the receptor proteins may evolve completely independently of the parent strain or species. The fact that there is a major distinction between the phylogenetic clades that contain the isotype I and II TbpBs, which differ substantially in size and properties, raises the question of whether there may be structural constraints on exchanges of sequence variation or not. An alternate Tf receptor has been identified in the bovine pathogens P. multocida and H. somni (Haemophilus somnus), which consists of a substantially smaller integral outer membrane protein from the TonB-dependent receptor family that does not have a lipoprotein coreceptor [44,45]. Sequence comparisons indicate that these receptors are more closely related to various heme or hemoglobin receptors, indicating that these likely arose through convergent evolution. In contrast to the classical TbpA protein, these TbpA2 proteins bind to the N- rather than C-lobe of Tf, and the presence of both receptor types in the same strain raises the question of whether this is simple redundancy or whether they may have advantages in different ecological niches. The TbpA2 protein would be predicted to have the same core structural features of TonB-dependent receptor proteins – 22-strand b-barrels with central plug domains – but would lack the large extracellular loops that distinguishes TbpA from siderophore receptors and other members of the TonB-dependent protein family. Thus, it will be interesting to determine whether TbpA2 also induces a conformational change in the Tf N-lobe that results in separation of the domains, www.futuremedicine.com
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and how this is accomplished. Since annotation of genomes is based on sequence comparisons with known proteins, it is unknown whether there are A2-type receptors or additional types of Tf receptors in other Gram-negative species, and whether alternate mechanisms of iron removal and transport are utilized. The bacterial Lf receptors were originally identified in important Gram-negative pathogens of humans from the Neisseriaceae and Moraxellaceae families [12,20,46], and are notably absent from the Pasteurellaceae, considering the prevalence of Tf receptors in species from this family. As with the Tf receptors, there is substantially greater sequence variation in the LbpB relative to the LbpA protein, likely a reflection of surface accessibility. While structural data are currently not available for the complete LbpB structure, the levels of sequence variation are similar to those of TbpB, with the exception being in the large negatively charged region [46]. In spite of the extensive sequence variation in the negatively charged regions based on conventional sequence comparison algorithms, there may be conserved features capable of inducing cross-reactive antibodies that may provide the selective pressures for its susceptibility to cleavage by NalP. Considering that the Lf receptors not only provide the ability to acquire iron from Lf, but also provide protection from cationic antimicrobial peptides [20], it seems surprising that these receptors are not present in bacteria from the Pasteurellaceae that reside in a similar niche. Perhaps these species have other mechanisms for protection from cationic antimicrobial peptides, but it may also indicate that they reside in a specific niche that differs from that occupied by the bacteria that possess Lf receptors. Future perspective
The recent success colonizing transgenic mice that express the human CEACAM1 receptor with N. meningitidis [47] provides an avenue to further explore the various factors influencing colonization of the upper respiratory tract. In the human CEACAM1 mouse, the provision of exogenous hTf results in colonization by N. meningitidis for periods of up to 10 days and it is anticipated that expression of hTf or Lf in place of the mouse proteins will result in extended colonization without provision of exogenous iron sources. Colonization studies in human CEACAM1–Tf–Lf mice with a set of isogenic mutant strains of N. meningiti dis will provide the opportunity to explore the relative importance of TbpA, TbpB, LbpA and LbpB in colonization of the upper respiratory tract. Inclusion of a mutant LbpB that lacks the 1582
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clusters of negatively charged amino acids in the C-lobe region [48] will provide the opportunity to tease out the impact of LbpB on iron acquisition and protection against cationic peptides during the colonization process. The humanized mouse line should also be suitable for establishing a colonization model for N. gonorrhoeae in the female genitourinary tract, and a similar set of mutant strains can be used to explore the role of the receptor proteins and negatively charged regions of LbpB in colonization of the genitourinary tract. The association of CEACAM1-dependent transcytosis of N. meningitidis with successful colonization of the CEACAM1 transgenic mice [47], which is also anticipated for N. gonorrhoeae, would mean that dependence upon TbpA, TbpB, LbpA and LbpB may be due to accessing Tf and Lf in the submucosal space. Thus, colonization does not necessarily indicate that Tf and Lf are available on the mucosal surface. The interaction of nontypeable H. influenzae and some strains of M. catarrhalis with human CEACAM1 and the demonstrated dependence on a CEACAM1 homolog for colonization of chinchillas by nontypeable H. influenzae [49,50] suggests that human CEACAM1–Tf–Lf transgenic mice may provide a colonization model for these bacterial species as well. Since the adherence of M. catarrhalis and nontypeable H. influ enzae to CEACAM1 is not known to be associated with transcytosis across epithelial cell layers, the use of isogenic strains of these species may ultimately provide evidence for access to Tf and Lf on the mucosal surface. The study of porcine and bovine pathogens provides surrogate host– pathogen systems capable of addressing similar questions, with the advantage of involving the natural host, thus eliminating concerns about additional host–pathogen interactions that have not been considered. Results from the surrogate porcine host–pathogen system [15] is our only real evidence to date supporting the importance of TbpB in iron acquisition in vivo, which we currently presume also applies in humans. Continued studies with surrogate host–pathogen systems will strengthen extrapolations to the human host, where opportunities for direct experimentation are very limited. However, the inherent cost of large animal experiments and the limited selection of nonhuman pathogens with both Tf and Lf receptors suggest that the study of mutants of N. meningitidis, N. gonor rhoeae and M. catarrhalis in humanized transgenic mice may be our best opportunity to explore the relative availability of Tf and Lf on mucosal surfaces. future science group
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Although the recently solved structures of the Tf–TbpA and Tf–TbpB complexes [27,28] have provided insights into the iron acquisition process, our understanding of the bacterial Tf receptor iron uptake process is still incomplete. In the absence of high-resolution structural information for TbpA not complexed with Tf or for the ternary Tf–TbpB–TbpA complex, details of the process of complex formation are still lacking, and the processes of dissolution of the ternary complex after removal of iron from Tf are still speculative. Structures of TbpA alone, structures of TbpB–Tf–TbpA complexes with TonB, FbpA or other components may be necessary to gain a more complete picture of the stages of the iron acquisition process. Just as a more complete understanding of the mechanism of transport by bacterial ABC transporters was achieved by acquiring structural information from a variety of different transport complexes [51], there should be continued pursuit of structural information of Tf and Lf receptors. The structure of the Tf–TbpA complex coupled with steered molecular dynamics provide a reasonable description of how the TonB interaction could provide a potential path for transport of iron across the membrane [28]. The conserved nature of the EIEYE motif in
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all TbpAs and LbpAs to date suggests that the mechanism for iron transport across the membrane by this class of proteins is conserved. The absence of this specific motif in the TbpA2 receptor protein does not preclude a similar mechanism being involved, but highlights the need to pursue structural studies with this class of receptor proteins. The lack of large extracellular loops in TbpA2 that could mediate Tf binding as they do in TbpA and the binding of TbpA2 to the N-lobe in lieu of the C-lobe indicates that the mechanism of iron removal and transport may be quite distinct in this class of Tf receptors. Although the TbpA2 receptor has only been found in bovine pathogens to date, a combination of bioinformatics and functional approaches may reveal homologs in other host species. Since the iron-loaded status of the Tf present in the mucosal niche is unknown (apo, holo, monoferric N-lobe or monoferric C-lobe), whether the preference for iron being present in the N- or C-lobe has any ecological significance is uncertain. In attempting to explain the specific cleavage of LbpB by NalP, it has been suggested that this receptor release provides a form of immune evasion that would reduce the bactericidal effectiveness of anti-LbpB antibodies [37]. This infers
Executive summary Role of bacterial receptors for transferrin & lactoferrin in colonization & infection Although there are extensive studies on iron homeostasis and its regulation, the mechanisms involved in distribution of transferrin (Tf) within the body and onto mucosal surfaces are not fully understood. Lactoferrin (Lf) is produced by glandular tissue and in neutrophil granules, but it should not be assumed to be the dominant iron-binding glycoprotein on all mucosal surfaces. Direct assessment of the availability of Tf and Lf on mucosal surfaces is challenging. The dependence on Tf and Lf receptors by some bacteria for colonization and survival provides indirect evidence for the presence of Tf and Lf. Microbes can influence the availability of Tf and Lf on mucosal surfaces. Structural & functional features of Tf & Lf receptors Crystallographic data of the TbpA–Tf and TbpB–Tf complexes can now be used in conjunction with biochemical data to provide a more accurate model of TbpA–Tf–TbpB, including the role of the anchor peptide in ternary complex formation. Further studies will be required to understand the means by which TbpB and TbpA cooperate in the iron acquisition process. The assumption that LbpB is structurally and functionally similar to TbpB may be misleading and neglects the role of NalP in the release of LbpB from the cell surface. The TbpA2 receptor is sufficiently distinct from TbpA, such that structural and functional studies will be required to determine the iron removal and uptake process. The significance of TbpA and TbpA2 binding to different lobes of Tf is not understood. Diversity & prevalence of Tf & Lf receptors Tf and Lf receptors were initially identified in important pathogens of humans and food production animals that normally reside in the upper respiratory or genitourinary tract, inferring an importance for pathogenesis of infection. The presence of the Tf and Lf receptors in commensal species suggests that the receptors are required for colonization and raises the question of whether they are associated with a particular niche. It is currently unknown whether the TbpA2 type of Tf receptor is restricted to pathogens of ruminant hosts and whether it provides a selective advantage in specific niches on the host mucosal surfaces.
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that LbpB is more immunogenic or produces more cross-reactive antibodies than TbpB due to the presence of the large negatively charged regions and is not as essential for the iron acquisition process. Further studies should be able to directly address these issues. It will be interesting to determine whether LbpB from M. catarrhalis is similarly released from the cell surface by a functional homolog of NalP, as the same selective forces should be present. References
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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Established the role of transferrin (Tf) and lactoferrin receptors in survival and initiation of disease in a human gonococcal infection model, and that Tf was present at higher levels on the mucosal surface (based on urinary levels) than lactoferrin prior to challenge, contrary to prior dogma.
TbpA residues required for transferrin–iron utilization by Neisseria gonorrhoeae. Infect. Immun. 76(5), 1960–1969 (2008). 23. Moraes TF, Yu R-H, Strynadka NC,
Schryvers AB. Insights into the bacterial transferrin receptor: the structure of transferrin binding protein B from Actinobacillus pleuropneumoniae. Mol. Cell 35(4), 523–533 (2009). 24. Yang X, Yu RH, Calmettes C, Moraes TF,
Schryvers AB. The anchor peptide of transferrin binding protein B is required for interaction with transferrin binding protein A. J. Biol. Chem. 286(52), 45165–45173 (2011).
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transferrin binding proteins are virulence factors in Actinobacillus pleuropneumoniae serotype 7 infection. FEMS Microbiol. Lett. 209(2), 283–287 (2002).
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microbiome in health and disease. Annu. Rev. Pathol. 7, 99–122 (2012). 17. Tan S, Noto JM, Romero-Gallo J, Peek RM
Jr, Amieva MR. Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog. 7(5), e1002050 (2011). nn
Interesting study demonstrating how bacteria can manipulate host Tf trafficking to deliver iron to the epithelial surface.
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Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria. FEMS Microbiol. Rev. 35, 498–514 (2011). 19. Marri PR, Paniscus M, Weyand NJ et al.
First to demonstrate a function for the anchor peptide of a lipoprotein in protein–protein interactions, which supports the view that LbpB is not involved in iron acquisition since NalP cleavage would abrogate the LbpB–LbpA interaction.
25. Calmettes C, Yu R-H, Silva LP et al.
Structural variations within the transferrin binding site on transferrin binding protein, TbpB. J. Biol. Chem. 286, 12683–12692 (2011). nn
First structure of a complex of TbpB and Tf, providing insights into function and indicating that conformational changes in Tf will be required for complex formation with TbpA.
26. Silva LP, Yu R, Calmettes C et al. Conserved
Genome sequencing reveals widespread virulence gene exchange among human
interaction between transferrin and transferrin-binding proteins from porcine
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pathogens. J. Biol. Chem. 286(24), 21353–21360 (2011).
36. Bonnah RA, Yu R-H, Schryvers AB.
Moraes TF. The structural basis of transferrin iron sequestration by transferrin binding protein B. Nat. Struct. Mol. Biol. 19(3), 358–360 (2012).
37. Roussel-Jazede V, Jongerius I, Bos MP,
basis for iron piracy by pathogenic Neisseria. Nature 483, 53–58 (2012). First reported structure of a TbpA–Tf complex and proposed model of a ternary complex. Provides a description of the proposed mechanism of iron removal and transport.
29. Schryvers AB, Stojiljkovic I. Iron acquisition
systems in the pathogenic Neisseria. Mol. Microbiol. 32, 1117–1123 (1999). 30. Silburt C, Roulhac P, Weaver K et al.
Hijacking transferrin bound iron: protein–receptor interactions involved in iron transport in N. gonorrhoeae. Metallomics 1(1), 249–255 (2009). 31. Khan AG, Shouldice SR, Kirby SM, Yu R-H,
Tari LW, Schryvers AB. High affinity binding by the periplasmic iron binding protein from Haemophilus influenzae is required for acquiring iron from transferrin. Biochem. J. 404(2), 217–225 (2007). 32. Silva LP, Yu RH, Calmettes C et al. Steric and
allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin. Biochem. J. 444, 189–197 (2012). 33. Noinaj N, Cornelissen CN, Buchanan SK.
Structural insight into the lactoferrin receptors from pathogenic Neisseria. J. Struct. Biol. 184(1), 83–92 (2013). 34. Wong H, Schryvers AB. Bacterial lactoferrin
binding protein a binds to both domains of the human lactoferrin C-lobe. Microbiology 149, 1729–1737 (2003). 35. Arutyunova E, Brooks CL, Beddek A, Mak
MW, Schryvers AB, Lemieux MJ. Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis. Biochem. Cell Biol. 90(3), 351–361 (2012).
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Tommassen J, van Ulsen P. NalP-mediated proteolytic release of lactoferrin-binding protein B from the meningococcal cell surface. Infect. Immun. 78(7), 3083–3089 (2010).
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Demonstrates the ‘specific’ release of LbpB from the surface of Neisseria meningitidis by NalP and suggests that this may be part of an immune evasion mechanism.
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AB. Patterns of sequence variation with the Neisseria meningitidis lactoferrin-binding proteins. Biochem. Cell Biol. 90(3), 339–350 (2012). 47. Johswich KO, McCaw SE, Islam E et al.
In vivo adaptation and persistence of Neisseria meningitidis within the nasopharyngeal mucosa. PLoS Pathog. 9(7), e1003509 (2013).
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E et al. Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival. PLoS Pathog. 7(5), e1002027 (2011).
Important reference demonstrating the first colonization model for N. meningitidis using humanized mice expressing the human CEACAM receptor. This opens the door to future studies with more fully humanized mice that could explore the roles of Tbps and Lbps in the colonization of N. meningitidis.
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and phenotypic modifications of Neisseria meningitidis after an accidental human passage. PLoS ONE 6(2), e17145 (2011). 39. Echenique-Rivera H, Muzzi A, Del Tordello
WM, Deal CD, Jerse AE. Experimental gonococcal infection in male volunteers: cumulative experience with Neisseria gonorrhoeae strains FA1090 and MS11mkC. Front. Microbiol. 2, 123 (2011). 41. Harrison OB, Maiden MC, Rokbi B.
Distribution of transferrin binding protein B gene (tbpB) variants among Neisseria species. BMC Microbiol. 8, 66 (2008). 42. Zhang Y, Heidrich N, Ampattu BJ et al.
Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell 50(4), 488–503 (2013). 43. Ambur OH, Frye SA, Nilsen M, Hovland E,
Tonjum T. Restriction and sequence alterations affect DNA uptake sequencedependent transformation in Neisseria meningitidis. PLoS ONE 7(7), e39742 (2012). 44. Ogunnariwo JA, Schryvers AB.
Characterization of a novel transferrin receptor in bovine strains of Pasteurella multocida. J. Bacteriol. 183(3), 890–896 (2001).
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Fernandez S, McGillivary G, Bakaletz LO. A carcinoembryonic antigen-related cell adhesion molecule 1 homologue plays a pivotal role in nontypeable Haemophilus influenzae colonization of the chinchilla nasopharynx via the outer membrane protein P5-homologous adhesin. Infect. Immun. 76(1), 48–55 (2008). 50. Brooks MJ, Sedillo JL, Wagner N et al.
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transporters: the power to change. Nat. Rev. Mol. Cell. Biol. 10(3), 218–227 (2009).
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Bacterial receptors for host transferrin and lactoferrin: molecular mechanisms and role in host–microbe interactions Ari Morgenthau1, Anastassia Pogoutse2, Paul Adamiak1, Trevor F Moraes2 & Anthony B Schryvers*1,3 Department of Microbiology, Immunology & Infectious Diseases, Health Sciences Centre, 3330 Hospital Drive Northwest Calgary, Alberta, T2N 4N1, Canada 2 Department of Biochemistry, Medical Sciences Building, King’s College Circle, Toronto, Ontario, M5S 1A8, Canada 3 Department of Biochemistry & Molecular Biology, Health Sciences Centre, 3330 Hospital Drive Northwest Calgary, Alberta, T2N 4N1, Canada *Author for correspondence: Tel.: +1 403 220 3703 n Fax: +1 403 210 9747 n [email protected] 1
Iron homeostasis in the mammalian host limits the availability of iron to invading pathogens and is thought to restrict iron availability for microbes inhabiting mucosal surfaces. The presence of surface receptors for the host iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) in globally important Gramnegative bacterial pathogens of humans and food production animals suggests that Tf and Lf are important sources of iron in the upper respiratory or genitourinary tracts, where they exclusively reside. Lf receptors have the additional function of protecting against host cationic antimicrobial peptides, suggesting that the bacteria expressing these receptors reside in a niche where exposure is likely. In this review we compare Tf and Lf receptors with respect to their structural and functional features, their role in colonization and infection, and their distribution among pathogenic and commensal bacteria. Role of bacterial receptors for transferrin & lactoferrin in colonization & infection
Due to the critical role that iron plays in mammalian metabolism and its potential toxicity, mammals have developed effective systems for handling iron and regulating levels of available iron [1]. The host iron-binding protein transferrin (Tf) plays a central role in systemic iron homeostasis by shuttling iron beween cells that supply iron to those that require it. Dietary iron acquired from gut enterocytes or recycled erythrocyte iron processed by macrophages or hepatocytes is loaded onto Tf through the combined action of the ferrous iron transporter ferroportin, which exports iron from the cell, and a ferroxidase (hephestin or ceruloplasmin), which converts ferrous iron to ferric ion in order to be bound by Tf. These processes are regulated by hepcidin, a key regulatory molecule in iron homeostasis, which influences the stability of ferroportin [2]. The iron-loaded (holo)-Tf supplies iron to various cells throughout the body via a process initiated by the binding of holo‑Tf to the human Tf (hTf ) receptor TFR1. The bound holo‑Tf is internalized by receptor-mediated endocytosis, and iron removal is facilitated by the acidic environment of the endosome. The receptor-bound iron-free apo-Tf is returned to 10.2217/FMB.13.125 © 2013 Future Medicine Ltd
the surface by fusion of the receptor-containing vesicle with the cytoplasmic membrane, where the apo‑Tf dissociates from the TFR1 receptor at the physiological pH of the extracellular milieu. Tf can also be endocytosed upon binding to TFR2 (HFE3) in some tissue types, but iron delivery is not thought to be the primary function of this receptor. In normal plasma, Tf is only approximately 30% iron-saturated and serves to readily sequester any nonheme iron that is released into the extracellular environment within the body [3]. The iron sequestration by Tf reduces the levels of free iron below that required for bacterial growth [4]. The structurally related iron-binding glycoprotein lactoferrin (Lf) serves a similar role, but is thought to primarily act on mucosal surfaces and sites of inflammation [5]. Lf has a higher affinity for iron than Tf, particularly at the lower pH at sites of inflammation. Lf is secreted in its apo form by epithelial cells (Figure 1) into most exocrine secretions and is released from secretory granules of neutrophils at sites of inflammation. Proteolytic cleavage of Lf leads to the release of a cationic antimicrobial peptide, lactoferricin, derived from the N-terminus. Lf is present at very low levels in tissue interstitial fluids, but can be found at high levels at sites of inflammation. Future Microbiol. (2013) 8(12), 1575–1585
Keywords bacterial receptors n iron acquisition n iron homeostasis n lactoferrin n transferrin n
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Neutrophil Neisseria
Lf Tf Mucosal layer ?
Bleeding?
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Endothelial cells Neutrophil
Figure 1. Availability of transferrin and lactoferrin on mucosal surfaces. Lf is released from secondary granules of neutrophils at sites of inflammation and can provide Lf on the mucosal surface due to neutrophil migration. Lf is also produced directly by epithelial glandular cells for secretion onto mucosal surfaces. Tf in serum could be released onto mucosal surfaces by episodic bleeding, but the route for delivering Tf to the mucosal surface from the submucosal space has not been determined. Tf in the submucosal space can be taken in by receptor-mediated endocytosis to provide iron to the epithelial cells, and some bacteria have been shown to hijack the recycling pathway to deliver Tf to the epithelial surface (yellow-colored bacterium). Lf: Lactoferrin; Tf: Transferrin.
Similarly, Lf is normally found at low levels in serum (0.4–2 mg/l), but can increase up to 200 mg/l during sepsis [6]. Owing to this, there is a generally held view that Tf is the major iron-binding protein within the body and Lf is the dominant iron-binding protein on mucosal surfaces. Gram-negative bacteria from the families Neisseriaceae, Moraxellaceae and Pasteurellaceae primarily reside in the upper respiratory or genitourinary tract. In humans, they constitute major causes of meningitis (Haemophilus influen zae and Neisseria meningitidis), upper and lower respiratory infections (Moraxella catarrhalis and H. influenzae) and genitourinary infections (Neisseria gonorrhoeae) [7–9]. In cattle, they are the primary cause of the bovine respiratory disease complex (Mannheimia haemolytica, Pasteurella multocida and Histophilus somni) plaguing cattle feedlots [10]. Members of Pasteurellaceae (Actinobacillus pleuropneumoniae, Haemophilus parasuis, Actinobacillus suis and P. multocida) predominantly affect the pig industry [11]. A common adaptation that these pathogens share for survival in the respiratory or genitourinary tract of their host is the possession of surface 1576
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receptors that directly bind Tf or Lf to acquire iron for growth [12]. The receptors are exquisitely specific for Tf and Lf from their mammalian host [13], corresponding to the restricted host range of these pathogens. Although the Tf receptors would clearly be advantageous for survival during invasive infection, their ubiquitous presence argues for the availability of Tf on the mucosal surface where the bacteria possessing them primarily reside. However, the mechanisms for the transport of Tf to the mucosal surface have not been established (Figure 1). Gonococcal infection studies in male volunteers demonstrated that survival of the bacteria was dependent upon the presence of either a Tf or Lf receptor, and led to the observation that the mucosal levels of Tf (inferred by measurements of urinary levels) exceed that of Lf prior to challenge [14]. Similarly, the requirement of both protein components of the bacterial Tf receptor for survival of A. pleuro pneumoniae in an aerosol challenge experiment [15] suggests that Tf is an important iron source in the upper respiratory tract of pigs. In contrast to the GI tract where exogenous sources of iron would readily be available, the available iron on the upper respiratory and genitourinary future science group
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tracts would be primarily derived from the host, which may explain why Tf and Lf receptors have only been observed in bacteria colonizing these surfaces. The challenges in obtaining accurate measure ments of the levels of Tf and Lf on mucosal surfaces have resulted in a relative lack of reliable information regarding Tf and Lf on the mucosal surfaces of the upper respiratory and genito urinary tracts. The Tf and Lf receptor-dependent growth of bacteria provides indirect evidence for the availability of these proteins and, at present, is perhaps one of our best sources of information regarding whether there are adequate levels of these proteins to support microbial growth. The mucosal surface is a critical interface between the host and the resident microbiome [16] and there is an increasing recognition of the complex interplay between the host and microbial community. Recently, it has been demonstrated that Helicobacter pylori is able to modulate the Tf recycling pathway in host epithelial cells with CagA and VacA, resulting in transport of submucosal Tf to the epithelial surface [17]. Thus, it will be important to consider the impact of the resident microbiome when evaluating the availability of Tf and Lf on various mucosal surfaces (F igure 1). An additional consideration is that some bacteria are capable of attaching to host surface proteins to mediate transcytosis across epithelial cells, thus they may be able to reside in the submucosal space [18]. The pathogenic Neis seria spp. possess phase-variable Opa adhesion proteins that mediate this process through interaction with CEACAM1 receptors expressed on
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epithelial cells. Tf and Lf receptors and opacityassociated proteins are also found in some of the ‘nonpathogenic’ Neisseria spp. [19], which raises interesting questions regarding whether accessing a unique ecological niche (the submucosal space) and utilization of Tf and Lf could be viewed as a first step towards developing pathogenicity. In other words, some commensal species have attributes shared with the pathogenic species and may be capable of causing disease if additional attributes are acquired. The recent demonstration that one of the Lf receptor proteins, LbpB, protects against the cationic antimicrobial peptide lactoferricin [20] indicates that enhanced survival of bacteria that possess Lf receptors may be due to more than one factor (Box 1). Structural & functional features of Tf & Lf receptors
The canonical Tf receptor in Gram-negative bacteria consists of two proteins that are each capable of binding Tf independently: TbpA and TbpB (Figure 2). TbpB has a strong preference for binding the iron-loaded (holo) form of Tf, whereas TbpA binds both apo- and holo‑Tf with equal affinity. As TbpB is a surfaceexposed lipoprotein, the selective capture of holo‑Tf has been proposed to serve the role of delivering holo‑Tf to the integral outer membrane protein TbpA (Figure 2). With the notable exception of N. meningitidis strain B16B6 [21] or in N. gonorrhoeae TbpA mutants [22], growth on Tf as an iron source under laboratory conditions does not require the presence of TbpB.
Box 1. Availability of transferrin and lactoferrin on mucosal surfaces. Transferrin (Tf) is the predominant iron-containing extracellular protein within the body; therefore, Tf receptors should play a critical role during infection Tf receptors are required for colonization/infection of the male genitourinary tract and the significant levels of measured urinary Tf prior to challenge infers normal availability on the mucosal surface Tf receptors are required for colonization/infection of the porcine upper respiratory tract The mechanism for transport of Tf to the mucosal surface is not known (Figure 1) A mechanism by which bacteria hijack the Tf recycling pathway to bring Tf to the epithelial surface has been described for Helicobacter pylori, raising the spectre of microbial modulation of Tf availability on other mucosal surfaces (Figure 1) Normal physiological levels of lactoferrin (Lf) in serum are low, but due to the inflammatory response during invasive infection, Lf levels may substantially increase, particularly in localized areas Mechanisms for Lf export to the mucosal surface are known (Figure 1) , but the normal levels may be relatively low, as observed in the male genitourinary tract Lf receptors may play an important role during colonization, but the relative importance for iron acquisition and protection from cationic antimicrobial peptides is uncertain Assumptions that Lf receptors were primarily important on mucosal surfaces need to be revisited and the relative role in iron acquisition and protection from cationic antimicrobial peptides needs to be assessed
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Lipo-oligosaccharide Outer membrane FbpA ExbD TonB ExbB
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Fe FbpB
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Figure 2. Iron uptake mediated by transferrin and lactoferrin receptors. (A) Tf and Lf receptors consist of an integral outer membrane protein (TbpA or LbpA) and a membrane-anchored lipoprotein (TbpB or LbpB). NalP is capable of selectively cleaving the anchor peptide of LbpB. (B) Membrane-anchored TbpB captures iron-loaded Tf with its N-lobe, whereas released LbpB binds to Lf through the negatively charged region in the C-lobe. (C) TbpB delivers Tf to TbpA to initiate the removal and transport of iron to the periplasmic iron-binding protein FbpA. The released LbpB would not facilitate transfer of Lf to LbpA. Lf: Lactoferrin; Tf: Transferrin.
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Thus, the role of TbpB in delivering holo‑Tf to TbpA is thought to only be essential under the more stringent growth conditions encountered in vivo. The first published structure of TbpB from the porcine pathogen A. pleuropneumoniae demonstrated that TbpB is made up of two structurally similar lobes, termed the N-lobe and the C-lobe, which are positioned orthogonally with respect to each other and connected by a flexible linker [23]. Each of the TbpB lobes contains two domains, a b-barrel domain that sits parallel to a handle domain. TbpB also contains an anchor peptide that attaches to the lipid moiety responsible for tethering the protein to the membrane (Figure 2). Only the last (C-terminal) 17 amino acids of the anchor peptide region were resolved in the structure and shown to be intimately associated with the C-terminal lobe, effectively wrapping around the external surface of the C-terminal lobe. The purpose of this unusual association of the anchor peptide with the C-terminal lobe is unknown, but it has been speculated that modulation of the interaction upon binding of holo‑Tf by TbpB could promote interaction of TbpB with TbpA (Figures 2C & 3) [24]. A preliminary model for the complex between TbpB and Tf, involving the TbpB N-lobe and porcine Tf C-lobe, was generated using computational docking and supported by site-directed mutagenesis of the residues from the predicted binding surface of TbpB [23]. Improved models for the interaction of TbpB with Tf were obtained with additional TbpB structures by combining experimental data from hydrogen–deuterium exchange coupled with mass spectrometry experiments and binding assays performed with sitedirected mutants of the TbpBs [25,26]. The development and use of a program that incorporated constraints from the experimental data into the standard Rosetta docking algorithms resulted in models of the complex that involved the same interface, but differed in the orientation of Tf relative to TbpB by 180°. The structure of the complex between N. meningitidis TbpB and hTf determined by protein crystallography confirms this latter orientation and demonstrates that the region of Tfs recognized by TbpBs from diverse strains and species are essentially the same [27]. Interestingly, the binding interface of TbpB on Tf partially overlaps with the interface recognized by the mammalian Tf receptor, indicating that simultaneous binding by both receptors is not possible [3]. The Tf–TbpB structure indicates that the Tf C-lobe is in the closed, iron-loaded conformation, strongly suggesting that TbpB future science group
Review
locks Tf in the iron-loaded form by creating an environment that limits the charge repulsion between two key residues in the C-lobe cleft. The structural results are consistent with the role of TbpB delivering the iron-loaded form of Tf to TbpA at the bacterial cell surface (Figure 2). The recently solved structure of a complex of N. meningitidis TbpA and hTf [28] has provided considerable insights into the iron removal and transport process. TbpA contains a 22-stranded b-barrel encircling a plug domain with Tf bound through the long loops on the extracellular side of the protein. The structure of the TbpA–Tf complex shows the Tf C-lobe to be in a semiopen conformation, with no iron in its binding site. A portion of loop 3 of TbpA, termed the ‘helix finger’, is inserted into the cleft between the C1 and C2 subdomains of the Tf C-lobe, along with a loop extending from the plug domain. Owing to this, it is thought that the conformational change in the Tf C-lobe that occurs upon binding to TbpA lowers the affinity for binding iron, as was originally proposed [29]. Steered molecular dynamics provides a reasonable description of how interaction with TonB in the periplasm could provide a potential path for
TbpB
hTf
TbpB Anchor peptide
Outer membrane
TbpA
Figure 3. Model of the TbpB–transferrin–TbpA ternary complex. An updated model of the TbpB–transferrin–TbpA complex using structural information from the crystallized TbpB–transferrin complex [20] . In this model, there is a 180° shift in the orientation of TbpB relative to hTf compared with the published model [28] , which positions the junction between the anchoring peptide and N-lobe handle domain, closer to the membrane surface. This orientation provides greater opportunity for interactions between the TbpB-anchoring peptide and TbpA. The arrow signifies a potential interaction between the anchoring peptide of TbpA and TbpB. hTf: Human transferrin. Data taken from [20] .
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transport of iron across the membrane, utilizing the conserved EIEYE motif [28]. However, this does not provide an explanation of what is driving the movement across the membrane, nor how otherwise insoluble ferric iron could be released at the periplasmic face. The possibility that this process is driven by capture of the ferric iron by the apo form of the periplasmic iron binding protein FbpA (Figure 2) through intimate interactions with TbpA is supported by prior stability of unpurified proteins from rates of H/D exchange studies [30] and site-directed mutagenesis studies with FbpA [31]. While the TbpB–Tf and TbpA–Tf structures [27,28] provide insight into how TbpA and TbpB interact with Tf individually, they provide little insight into how TbpA and TbpB work in concert to utilize Tf-bound iron (Box 2). A ternary TbpB–Tf–TbpA complex can be formed from either a TbpA–Tf or a TbpB–Tf complex, but requires the presence of the intact TbpB anchor peptide [24], which is necessary, but not sufficient, for binding. The TbpA and TbpB footprints on Tf are distinct [27,28,32], but TbpB and TbpA bind to the Tf C-lobe in the closed and partially open conformation, respectively, indicating that the TbpB–Tf interaction needs to be altered during the ‘hand-off’ to TbpA. A model for the ternary Tf–TbpA–TbpB complex has been developed by fitting structures of TbpB–Tf and TbpA–Tf complexes into small angle x-ray scattering (SAXS) and negative-stain electron microscopy data obtained from purified ternary complexes [28]. However, the authors did not have the crystallographic data for the N. menin gitidis TbpB–hTf complex at the time, thus they used the structural models of the complex of porcine Tf and A. pleuropneumoniae TbpB derived from a computational docking approach [23] as a template for the Tf–TbpB complex that was fitted into the envelope generated by the SAXS and electron microscopy data. The orientation of
hTf relative to TbpB in the model derived from the SAXS and molecular docking does not agree with the orientation shown in the Tf–TbpB structure obtained via x-ray crystallog raphy [27]. Thus, we have generated a new model of the complex using structural data from the TbpA–Tf and TbpB–Tf complexes (Figure 3). In this model, the alternate orientation of Tf relative to TbpB positions the anchor peptide in a more favorable orientation for interactions with TbpA. Clearly, additional experimental studies need to be directed towards determining the true nature of the ternary complex. In the absence of structural information for intact LbpB, LbpA and complexes with Lf, inferences regarding the structural and mechanistic features are primarily based on comparisons with the structural homologs from the Tf receptor system. It is a reasonable assumption that the LbpA structure will largely resemble TbpA and that the C-lobe of Lf will dock onto LbpA in a similar manner, as has been proposed [33], since prior experiments have established that LbpA binds to both domains of the Lf C-lobe [34]. The high degree of identity in the plug region and the conserved EIEYE motif suggests that the LbpA shares the mechanism for channel formation and iron transport across the membrane with TbpA. By contrast, inferences regarding the interaction between LbpB and Lf, and the role of LbpB in facilitating the iron acquisition process [33] are too speculative at present. To date, efforts to determine the structure of LbpBs have only yielded structures of the LbpB N-lobe [35] and there is no experimental evidence supporting the orthogonal association between the N-lobe and C-lobe that is observed in TbpB. In fact, the lack of structural data on full length LbpB may suggest a degree of flexibility between the N- and C-lobes. The assumption that the Cap region of the N-lobe of LbpB binds to the C-terminal lobe of Lf [33] ignores the obvious tendency for
Box 2. Structural and functional features of transferrin and lactoferrin receptors. The bacterial transferrin (Tf) receptor is composed of the TonB-dependent transporter TbpA and the lipid-anchored protein TbpB TbpB serves to capture holo‑Tf and transfer it to TbpA through the formation of a ternary complex involving the anchor peptide region The details of the process of ternary complex formation and the authentic structure of the ternary complex need to be determined The structure of the Tf–TbpA complex provides insights into the iron removal process and formation of a channel for transport of iron across the membrane, but the role of FbpA in the transport process is uncertain The structure of the TbpB–Tf complex may not be an appropriate model for the LbpB–lactoferrin interaction LbpB is cleaved by NalP, thus it is unlikely to serve a role equivalent to TbpB in iron acquisition
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the positively charged cationic region of the N1 domain of Lf to bind to the negatively charged regions in the C-lobe of LbpB (Figure 2) [20] and that the LbpB–Lf interaction is notably sensitive to pH and salt concentration [36]. This interaction is likely to be responsible for the observed role of LbpB in mediating protection against the cationic antimicrobial peptide lactoferricin [20]. The selective proteolytic release of LbpB from the bacterial surface by NalP [37] in conjunction with evidence for NalP expression in vivo [38,39] indicates that there will likely be excess released LbpB present in the host (Figure 2B & C). Proteo lysis of the anchor peptide makes it unlikely that it will be capable of facilitating the formation of the ternary complex, as has been observed in the mechanism for iron acquisition by the TbpA–TbpB receptor [24]. Thus, released LbpB would not facilitate the iron acquisition process (Figure 2), particularly if free Lf has a comparable affinity for LbpA as the Lbp–Lf complex. These findings all argue against LbpB playing a similar role in the iron acquisition process to TbpB. Thus, it is possible that LbpB has evolved from a protein with a similar role to TbpB into a protein whose primary function is related to protection from cationic peptides, and may have altered its original binding properties in the process. In this context, the observation that over half of the clinical gonococcal isolates are naturally deficient in Lf utilization while there was an observed selective advantage of having a functional lbp operon in the human gonococcal infection model [40] could be revisited from a different perspective. Prevalence & diversity of Tf & Lf receptors
The bacterial Tf receptors were originally identified in important Gram-negative pathogens of humans and food production animals from the Neisseriaceae, Pasteurellaceae and Moraxellaceae families [12]. These pathogens share a similar niche, colonizing the urogenital or respiratory tracts of their respective hosts, which serve as the only reservoirs for these highly host-adapted bacteria. The ubiquitous presence of the Tf receptors in clinical disease isolates from invasive infection argues that these components are important virulence factors by providing a sustained source of iron for growth during infection. However, the selective forces for retention of the Tf receptors are undoubtedly related to survival of the bacteria in the upper respiratory or genitourinary tracts, as was highlighted by the demonstration that the Tf receptor is critical for colonization in a human gonococcal challenge model [14]. The presence of Tf receptors in some commensal pathogenic future science group
Review
Neisseria spp. supports this hypothesis [41], but the absence of receptors from some commensal strains [19] begs the question of whether the Tf receptor provides a survival advantage primarily in a specialized niche that is only shared by a subset of the commensal bacteria or not. Overall there is greater sequence diversity in the TbpB than TbpA proteins, likely a reflection of the greater surface accessibility of the TbpB protein and a commensurate selective pressure for antigenic variation. Analysis of the distribution of tbpB genes within Neisseria spp. concluded that the phylogenetic clades assorted independently from species [41], suggesting that there may be a common gene pool available to closely related species sharing a particular niche. This is perhaps not surprising considering the common uptake sequences recognized by the natural transformation systems present in these species, although there clearly are various systems that influence horizontal exchange rates [42,43]. This observation also indicates that the receptor proteins may evolve completely independently of the parent strain or species. The fact that there is a major distinction between the phylogenetic clades that contain the isotype I and II TbpBs, which differ substantially in size and properties, raises the question of whether there may be structural constraints on exchanges of sequence variation or not. An alternate Tf receptor has been identified in the bovine pathogens P. multocida and H. somni (Haemophilus somnus), which consists of a substantially smaller integral outer membrane protein from the TonB-dependent receptor family that does not have a lipoprotein coreceptor [44,45]. Sequence comparisons indicate that these receptors are more closely related to various heme or hemoglobin receptors, indicating that these likely arose through convergent evolution. In contrast to the classical TbpA protein, these TbpA2 proteins bind to the N- rather than C-lobe of Tf, and the presence of both receptor types in the same strain raises the question of whether this is simple redundancy or whether they may have advantages in different ecological niches. The TbpA2 protein would be predicted to have the same core structural features of TonB-dependent receptor proteins – 22-strand b-barrels with central plug domains – but would lack the large extracellular loops that distinguishes TbpA from siderophore receptors and other members of the TonB-dependent protein family. Thus, it will be interesting to determine whether TbpA2 also induces a conformational change in the Tf N-lobe that results in separation of the domains, www.futuremedicine.com
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and how this is accomplished. Since annotation of genomes is based on sequence comparisons with known proteins, it is unknown whether there are A2-type receptors or additional types of Tf receptors in other Gram-negative species, and whether alternate mechanisms of iron removal and transport are utilized. The bacterial Lf receptors were originally identified in important Gram-negative pathogens of humans from the Neisseriaceae and Moraxellaceae families [12,20,46], and are notably absent from the Pasteurellaceae, considering the prevalence of Tf receptors in species from this family. As with the Tf receptors, there is substantially greater sequence variation in the LbpB relative to the LbpA protein, likely a reflection of surface accessibility. While structural data are currently not available for the complete LbpB structure, the levels of sequence variation are similar to those of TbpB, with the exception being in the large negatively charged region [46]. In spite of the extensive sequence variation in the negatively charged regions based on conventional sequence comparison algorithms, there may be conserved features capable of inducing cross-reactive antibodies that may provide the selective pressures for its susceptibility to cleavage by NalP. Considering that the Lf receptors not only provide the ability to acquire iron from Lf, but also provide protection from cationic antimicrobial peptides [20], it seems surprising that these receptors are not present in bacteria from the Pasteurellaceae that reside in a similar niche. Perhaps these species have other mechanisms for protection from cationic antimicrobial peptides, but it may also indicate that they reside in a specific niche that differs from that occupied by the bacteria that possess Lf receptors. Future perspective
The recent success colonizing transgenic mice that express the human CEACAM1 receptor with N. meningitidis [47] provides an avenue to further explore the various factors influencing colonization of the upper respiratory tract. In the human CEACAM1 mouse, the provision of exogenous hTf results in colonization by N. meningitidis for periods of up to 10 days and it is anticipated that expression of hTf or Lf in place of the mouse proteins will result in extended colonization without provision of exogenous iron sources. Colonization studies in human CEACAM1–Tf–Lf mice with a set of isogenic mutant strains of N. meningiti dis will provide the opportunity to explore the relative importance of TbpA, TbpB, LbpA and LbpB in colonization of the upper respiratory tract. Inclusion of a mutant LbpB that lacks the 1582
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clusters of negatively charged amino acids in the C-lobe region [48] will provide the opportunity to tease out the impact of LbpB on iron acquisition and protection against cationic peptides during the colonization process. The humanized mouse line should also be suitable for establishing a colonization model for N. gonorrhoeae in the female genitourinary tract, and a similar set of mutant strains can be used to explore the role of the receptor proteins and negatively charged regions of LbpB in colonization of the genitourinary tract. The association of CEACAM1-dependent transcytosis of N. meningitidis with successful colonization of the CEACAM1 transgenic mice [47], which is also anticipated for N. gonorrhoeae, would mean that dependence upon TbpA, TbpB, LbpA and LbpB may be due to accessing Tf and Lf in the submucosal space. Thus, colonization does not necessarily indicate that Tf and Lf are available on the mucosal surface. The interaction of nontypeable H. influenzae and some strains of M. catarrhalis with human CEACAM1 and the demonstrated dependence on a CEACAM1 homolog for colonization of chinchillas by nontypeable H. influenzae [49,50] suggests that human CEACAM1–Tf–Lf transgenic mice may provide a colonization model for these bacterial species as well. Since the adherence of M. catarrhalis and nontypeable H. influ enzae to CEACAM1 is not known to be associated with transcytosis across epithelial cell layers, the use of isogenic strains of these species may ultimately provide evidence for access to Tf and Lf on the mucosal surface. The study of porcine and bovine pathogens provides surrogate host– pathogen systems capable of addressing similar questions, with the advantage of involving the natural host, thus eliminating concerns about additional host–pathogen interactions that have not been considered. Results from the surrogate porcine host–pathogen system [15] is our only real evidence to date supporting the importance of TbpB in iron acquisition in vivo, which we currently presume also applies in humans. Continued studies with surrogate host–pathogen systems will strengthen extrapolations to the human host, where opportunities for direct experimentation are very limited. However, the inherent cost of large animal experiments and the limited selection of nonhuman pathogens with both Tf and Lf receptors suggest that the study of mutants of N. meningitidis, N. gonor rhoeae and M. catarrhalis in humanized transgenic mice may be our best opportunity to explore the relative availability of Tf and Lf on mucosal surfaces. future science group
Bacterial receptors for host transferrin & lactoferrin
Although the recently solved structures of the Tf–TbpA and Tf–TbpB complexes [27,28] have provided insights into the iron acquisition process, our understanding of the bacterial Tf receptor iron uptake process is still incomplete. In the absence of high-resolution structural information for TbpA not complexed with Tf or for the ternary Tf–TbpB–TbpA complex, details of the process of complex formation are still lacking, and the processes of dissolution of the ternary complex after removal of iron from Tf are still speculative. Structures of TbpA alone, structures of TbpB–Tf–TbpA complexes with TonB, FbpA or other components may be necessary to gain a more complete picture of the stages of the iron acquisition process. Just as a more complete understanding of the mechanism of transport by bacterial ABC transporters was achieved by acquiring structural information from a variety of different transport complexes [51], there should be continued pursuit of structural information of Tf and Lf receptors. The structure of the Tf–TbpA complex coupled with steered molecular dynamics provide a reasonable description of how the TonB interaction could provide a potential path for transport of iron across the membrane [28]. The conserved nature of the EIEYE motif in
Review
all TbpAs and LbpAs to date suggests that the mechanism for iron transport across the membrane by this class of proteins is conserved. The absence of this specific motif in the TbpA2 receptor protein does not preclude a similar mechanism being involved, but highlights the need to pursue structural studies with this class of receptor proteins. The lack of large extracellular loops in TbpA2 that could mediate Tf binding as they do in TbpA and the binding of TbpA2 to the N-lobe in lieu of the C-lobe indicates that the mechanism of iron removal and transport may be quite distinct in this class of Tf receptors. Although the TbpA2 receptor has only been found in bovine pathogens to date, a combination of bioinformatics and functional approaches may reveal homologs in other host species. Since the iron-loaded status of the Tf present in the mucosal niche is unknown (apo, holo, monoferric N-lobe or monoferric C-lobe), whether the preference for iron being present in the N- or C-lobe has any ecological significance is uncertain. In attempting to explain the specific cleavage of LbpB by NalP, it has been suggested that this receptor release provides a form of immune evasion that would reduce the bactericidal effectiveness of anti-LbpB antibodies [37]. This infers
Executive summary Role of bacterial receptors for transferrin & lactoferrin in colonization & infection Although there are extensive studies on iron homeostasis and its regulation, the mechanisms involved in distribution of transferrin (Tf) within the body and onto mucosal surfaces are not fully understood. Lactoferrin (Lf) is produced by glandular tissue and in neutrophil granules, but it should not be assumed to be the dominant iron-binding glycoprotein on all mucosal surfaces. Direct assessment of the availability of Tf and Lf on mucosal surfaces is challenging. The dependence on Tf and Lf receptors by some bacteria for colonization and survival provides indirect evidence for the presence of Tf and Lf. Microbes can influence the availability of Tf and Lf on mucosal surfaces. Structural & functional features of Tf & Lf receptors Crystallographic data of the TbpA–Tf and TbpB–Tf complexes can now be used in conjunction with biochemical data to provide a more accurate model of TbpA–Tf–TbpB, including the role of the anchor peptide in ternary complex formation. Further studies will be required to understand the means by which TbpB and TbpA cooperate in the iron acquisition process. The assumption that LbpB is structurally and functionally similar to TbpB may be misleading and neglects the role of NalP in the release of LbpB from the cell surface. The TbpA2 receptor is sufficiently distinct from TbpA, such that structural and functional studies will be required to determine the iron removal and uptake process. The significance of TbpA and TbpA2 binding to different lobes of Tf is not understood. Diversity & prevalence of Tf & Lf receptors Tf and Lf receptors were initially identified in important pathogens of humans and food production animals that normally reside in the upper respiratory or genitourinary tract, inferring an importance for pathogenesis of infection. The presence of the Tf and Lf receptors in commensal species suggests that the receptors are required for colonization and raises the question of whether they are associated with a particular niche. It is currently unknown whether the TbpA2 type of Tf receptor is restricted to pathogens of ruminant hosts and whether it provides a selective advantage in specific niches on the host mucosal surfaces.
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that LbpB is more immunogenic or produces more cross-reactive antibodies than TbpB due to the presence of the large negatively charged regions and is not as essential for the iron acquisition process. Further studies should be able to directly address these issues. It will be interesting to determine whether LbpB from M. catarrhalis is similarly released from the cell surface by a functional homolog of NalP, as the same selective forces should be present. References
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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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