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Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores
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Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores Cécile Wandersman* and Igor Stojiljkovic† The major mechanisms by which Gram-negative bacteria acquire heme from host heme-carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors. The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors. Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems. Addresses *Unité des Membranes Bactériennes, Institut Pasteur (CNRS URA 1300) 25 rue du Dr. Roux, 75724 Paris Cedex 15, France; e-mail: [email protected] † Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA; e-mail: [email protected] Current Opinion in Microbiology 2000, 3:215–220 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations Hb hemoglobin Hpt haptoglobin
Introduction Bacterial cells each contain 105 to 106 iron ions which are essential for many metabolic pathways. The low solubility of iron (III) salts at physiological pH in the presence of oxygen is a serious obstacle to microorganisms that require iron [1]. Microorganisms colonizing human hosts are confronted with another problem: 99.9% of total body iron (approx. 4 g) is intracellular and thus not readily available [2]. Furthermore, the extracellular iron found in human plasma and lymphatic fluids (a total of 3 mg) is tightly bound to transferrin. Small amounts of hemoglobin (Hb) (5–50 mg/L) are found in plasma as a result of spontaneous hemolysis. Free plasma Hb is bound by the circulating heterotetrameric glycoprotein, haptoglobin (Hpt), which delivers Hb dimers to the liver. Plasma albumin and hemopexin bind circulating heme and deliver it to the liver. Mucosal surfaces contain significant amounts of free heme and inorganic iron mostly complexed to lactoferrin and various metabolic byproducts [3]. Thus, human body fluids and surfaces contain sufficient amounts of inorganic, protein-bound and heme-associated iron to support bacterial growth. Therefore, for a bacterial pathogen to successfully colonize and establish in a mammalian host, it must be able to assimilate iron from (one or more of) these diverse sources of iron.
Iron assimilation Iron containing molecules are not transported across the outer membrane of Gram-negative bacteria by passive
diffusion: their extracellular concentrations are low and they are relatively large. Instead, these molecules are recognized and bound by specific receptors at the cell surface and actively transported against a concentration gradient into the bacterial periplasm. The iron capture systems can be classified in two groups: those that involve direct binding of iron or iron-containing proteins, and those that rely on the excretion of siderophores (low molecular weight iron ligands) and their receptors. Intact iron-loaded siderophores are taken up through the outer membrane, whereas iron-containing proteins are unloaded at the cell surface and only iron reaches the periplasm and is transported to the cytoplasm. Two siderophore-specific outer membrane receptors, FepA [4] and FhuA [5••], have been recently crystallised. Both are monomeric transmembrane β-barrels plugged on the periplasmic side by a amino-terminal cork-like domain of the protein. The activity of these receptors is dependent on a protein complex comprising two inner membrane proteins, ExbB and ExbD, and TonB, a proline-rich protein anchored to the cytoplasmic membrane via its amino terminus [6,7]. Experimental data have revealed direct interaction between TonB and the siderophore-specific outer membrane receptors [8]. Site-directed disulfide crosslinking demonstrated physical contact between a conserved amino-terminal region (the TonB-box) of the BtuB receptor and the TonB region around Gln160 [9]. Additional TonB and receptor domains must physically interact since a FhuA variant lacking its amino-terminal cork domain including the TonB-box was still able to take up siderophore in a TonB-dependent manner [10••]. The carboxy-terminal region of TonB is most probably involved in these interactions since carboxy-terminally truncated TonB constructs are not functional and are unable to interact physically with the FepA receptor [11].
Heme assimilation Just as in the case of intact iron-loaded siderophores, entire heme is taken up via outer membrane receptors and transported into the bacterial periplasm, whereas heme-containing proteins are unloaded at the cell surface and the heme is transported to the periplasm. Once in the periplasm, the heme is then transported by specific ABC protein-dependent periplasmic permeases through the inner membrane [12]. The fate of heme after entering the bacterial cytoplasm is not fully understood. However, at least one Gram-negative bacterium, Neisseriae, uses a heme oxygenase-like enzyme to acquire heme-iron [13]. Currently, twenty two different Gram-negative outer membrane proteins involved in the utilization of heme compounds are listed in the GeneBank data base. All
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share limited sequence identity (17% to 23%) with the outer membrane siderophore, transferrin and lactoferrin receptors, the amino terminus being the most conserved part of these proteins. Heme receptors have additional highly conserved domain (the FRAP/NPNL domain) [14••]. The overall structure of heme receptors is expected to be very similar to that of crystalized siderophore receptors. By analogy with iron uptake systems, we classify heme-uptake systems in two groups: first, those that involve direct binding of heme or heme-containing host proteins to specific outer membrane receptors, and second, those that rely on the secretion of hemophores (extracellular bacterial hemoproteins), which interact in the extracellular environment with the heme source and
present it to specific receptors (Figure 1). Here, we describe the different heme uptake systems, and focus on some important parallels and some significant differences with the systems for iron uptake.
Outer membrane receptors for heme and host hemoproteins
A first subgroup of outer membrane receptors includes the heme/Hb receptors of Yersinia, Escherichia coli and Shigella that share between 65% and 90% amino acid identity [15–18]. Studies on one member of this group, the Y. enterocolitica HemR, suggest that these receptors use many different heme containing-compounds as sources of iron [14••].
Figure 1
Gram-negative bacteria heme-uptake systems. (a) The HemR receptor of Y. enterocolitica. Heme/Hb binds the HemR receptor and heme is transported through the receptor channel in a TonB-dependent manner. See Figure 2 for more details. Other HemR-type receptors include ShuA from Shigella, ChuA from E. coli O157:H7, and HmuA from Y. pestis [15,16,18]. (b) The HmbR receptor of H. influenzae. The receptor is highly adapted to one or two heme–protein complexes. (c) The neisserial HpuAB receptor consists of two outer membrane proteins HpuA, a membrane lipoprotein, and HpuB, a Ton-dependent receptor. (d) Hemophores are secreted extracellularly where they bind
heme. The hemophere delivers the bound heme to a specific outer membrane receptor (HasR). The heme is transported into the cell by a TonB-dependent manner. The HasA family of hemophores are found in Y. pestis, P. aeruginosa, P. fluorescens and S. marcescens [35••]. The hemophore HxuA, which binds hemopexin, is found in H. influenzae [38]. The ABC protein is a membrane-associated ATPase belonging to the ATP-binding cassette protein superfamily [31]. The ABC protein is involved in heme transport from the periplasm to the cytoplasm. IM, inner membrane; OM, outer membrane; N, amino terminus; COOH, carboxyl terminus.
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Site-directed mutagenesis of the invariant histidine residue (His461) in the conserved FRAP/NPL domain in Y. enterocolitica HemR completely abolished the ability of the cell to feed on heme–protein complexes and severely impaired the use of free heme. Another highly conserved and functionally important histidine was identified at position 128 located in the putative cork-like domain of HemR. HemR receptor mutants in His128 were severely affected in the use of all heme sources, but still bind Hb–agarose to the outer membrane. These observations suggest that His128 and His461 participate in steps following the Hb–receptor contact. A hypothetical model (Figure 2) of HemR function postulates a TonB-dependent transfer of heme from the external His461 to the cork-positioned His128 such that heme is transported through the receptor channel [14••]. This transfer is accomplished by a change in heme-binding affinity of His461 and His128 that is generated by receptor–TonB interaction. As heme is buried within different hemoproteins with a very high affinity of association, it is unclear how it becomes available for interaction with HemR. The receptor may act as a surface chaperone and unfold the hemoproteins (see [19•]). However, HemR interacts with diverse hemoproteins that do not share significant similarity and no HemR–hemoprotein interactions have been shown biochemically, although they may be very weak and, therefore, not detectable by standard binding techniques. The second subgroup includes the heme receptors found in Vibrio, Neisseria and Haemophilus, including HutA, HmbR, HgbA, and Hpt–Hb receptors of H. influenzae [20–25]. These proteins are highly adapted to one or two heme–protein sources (Hb and/or Hpt–Hb complexes). They possess a high-affinity binding site for a particular heme–protein
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compound, and none of those tested allow growth on other heme–protein compounds. This suggests that these substrate-specific heme receptors directly recognize a protein carrier, whereas HemR and its homologs are able to recognize heme bound to diverse protein carriers.
The neisserial Hpt–Hb and Hb receptor, HpuAB, is a special case among the known heme receptors [26]. It consists of two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB). HpuB is TonB-dependent and similar to other single component TonB-dependent receptors (Figure 1). HpuA is an outer membrane lipoprotein. Both proteins are required for Hb, Hpt–Hb and apo-haptoglobin binding to whole cells. This bipartite hemoglobin receptor is very similar to the Neisseria transferrin (TbpAB) and lactoferrin (LbpAB) receptors [27]. Both components of the transferrin receptor are necessary for efficient transferrin binding; TbpA, the outer membrane TonB-dependent receptor, binds apo- and holo-transferrin similarily, whereas TbpB (the lipoprotein) discriminates between the loaded and unloaded substrate and binds ferrated transferrin with a 100 times higher affinity. The accessory lipoprotein, HpuA, may have a role very similar to that of TbpB. Unfortunately, it is not yet known whether HpuA and HpuB exhibit similar affinities for loaded and unloaded haptoglobin. Hemophore-dependent heme uptake
Hemophores (HasA) are small extracellular proteins produced by several Gram-negative bacteria [28–30]. They are secreted by ABC transporters, a process involving their carboxy-terminal secretion signal [31]. They form an independent family of heme-binding proteins without
Figure 2
A hypothetical mode of Y. enterocolitica HemR function. Heme binds external His461 and is transferred to the His128. This transfer is accomplished by a change in heme-binding affinity of His461 and His128 that is generated by a receptor–TonB interaction.
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homology to any other known proteins. The role of the hemophore is to bind heme and to deliver it to a specific outer membrane receptor, HasR. This receptor is essential for heme acquisition, whereas the hemophore is not, although it increases by a 100-fold the efficiency of the system for free or Hb-bound heme [32]. HasA is, however, essential for heme acquisition from other heme sources such as hemopexin and myoglobin (S Létoffé et al., unpublished data). By extracting heme from these sources, HasA supplies new heme substrate to HasR. Hence hemophores fulfill a function similar to that of siderophores: they are secreted to the extracellular medium and then return to the cell surface. They enhance the capacity of the system and broaden the spectrum of potential heme sources. It is also plausible but not demonstrated that they secure heme from other organisms. The Serratia marcescens HasA protein has been biochemically characterized. It exhibits a very high affinity for heme (Kd lower than 10–8 M), but does not undergo major structural changes upon heme binding [33]. The crystal structure of the heme–HasA complex has been resolved showing that the heme iron is highly exposed to the solvent and is bound to residues His32 and Tyr75 [34]. Both hemefree (apoprotein) and heme-loaded (holoprotein) HasA bind to HasR and they do so with similar apparent affinities (10–10 M), indicating direct protein–protein interactions between HasA and HasR [35••]. Apo-HasA bound to its outer membrane receptor, HasR, may either rapidly dissociate allowing a loaded molecule to bind or be able to bind heme present on holo-HasA. The HasA/HasR system is reminiscent of the Neisseria bipartite receptors HpuAB and TbpAB described above. HasA may have a function similar to that of the lipoproteins, HpuA and TbpB, enhancing the local heme concentration close to the receptor. Binding of the ligand-free form of the siderophore with an affinity similar to the loaded form has also been observed for a Pseudomonas aeruginosa siderophore pyoverdine [36•]. In this case, it is also not clear how the loaded form replaces the apo-siderophore and promotes iron transfer. The mechanism by which HasA extracts heme from hemoproteins is unknown. Clearly, heme transfer does not involve a stable complex between HasA and Hb [35••]. Heme capture from Hb by HasA might require either a transient protein complex or HasA to bind heme only when it spontaneously dissociates from hemoglobin. Another system involving hemophore has been described in H. influenzae. HxuA, which does not share sequence similarity with HasA, is secreted by a signal peptide-dependent pathway involving one outer membrane helper protein. HxuA is able to acquire heme from hemopexin and to return it to a specific receptor in the outer membrane [37,38].
The function of TonB in heme acquisition Various studies show the presence of highly conserved TonB proteins in all Gram-negative bacteria and suggest
the importance of TonB in heme acquisition processes. However, the available data concerning the role of TonB in heme uptake do not elucidate its molecular mechanism, despite the X-ray crystallography data obtained for two outer membrane siderophore receptors. TonB may also be involved in steps other than heme transport itself, in particular the binding of heme, hemoprotein and hemophore to their respective outer membrane receptors, and heme striping from heme carriers. In all studies done so far binding of hemoproteins did not depend on the presence of TonB ([35••]; M Baer et al., unpublished data). However, the kinetics of binding of hemoproteins to the receptors have not been studied and thus there is a possibility that parameters of binding, such as affinity and off/on rates, might be quite different between TonB+ and TonB– cells. Several lines of evidence indicate that the subsequent step of heme transfer from the heme carrier to the receptor might be TonB dependent. Data for diverse Hb receptors show that the unloading occurs only in TonB+ strains [39,40]. However, these experiments did not clearly dissociate the unloading step from the uptake step, which is TonB dependent. Heme uptake involves the receptors being unloaded of heme and thereby uptake generates new available sites. Previously, it was thought that TonB was unique and was shared by most of the iron uptake systems in each species. However, several recent studies have indicated the existence of TonB proteins that specialise in heme and hemoprotein uptake. In V. cholerae, two sets of tonB, exbB, and exbD genes have been identified [41••]. They have at least partially redundant functions since inactivation of each set did not result in a defect in iron or heme uptake. The tonB1 locus was shown to be part of the hut operon dedicated to heme uptake. DNA sequence data show the presence of such analogs also in Pseudomonas aeruginosa and S. marcescens ([42]; JM Ghigo et al., unpublished data). Redundancy of TonBs in these species is most likely to be evidence of a distant horizontal spread of heme-uptake operons between different microorganisms. Since receptor–TonB interactions are to some extent species-specific, a newly acquired heme uptake system can only be fully exploited if the heme receptor is accompanied by its cognate TonB protein. Further, an increase in the amount of TonB in the cell may make the transport of TonB-dependent substrates more efficient since different receptors normally compete for limiting amounts of TonB [43].
Concluding remarks Notable progress has been made during the past decade in the identification of bacterial surface receptors for hemecontaining compounds. These studies reveal that heme receptors, unlike siderophore receptors, have at least two distinct structural designs. The large number of different heme-containing compounds present in the biosphere was probably the major driving force behind the evolution of different types of heme receptors. Partial redundancy of
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heme systems in Gram-negative bacteria may be a consequence of the diversity of heme sources in the environment and an adaptation to rapidly changing amounts and varieties of hemoproteins encountered during various stages of infection. This redundancy together with the phase variation of receptor expression must also be a successful protection strategy against immune surveillance in the host [44–47]. Indeed, previous work has established that heme receptors are expressed in vivo during infection, and that the ability to use heme and Hb contributes to the colonization potential and virulence of pathogenic bacteria [21,48–51]. All heme receptors share significant amino acid sequence similarity with siderophore receptors indicating a common mechanism. However, heme receptors are confronted with more complicated task than siderophore receptors since they deal with heme tightly bound to diverse protein carriers. Currently, it is not clear how receptors extract heme from heme carrier proteins and hemophores. It is unlikely that recently discovered proteases, able to cleave Hb, are the answer to this problem [52,53]. Therefore, to unravel the mechanism of heme-receptor function will probably require deciphering the nature of interaction between receptors and heme-carrier proteins and defining the role of the TonB protein in these interactions.
Acknowledgements We thank Sylvie Létoffé and Jean Marc Ghigo for helpful discussions. The work in Igor Skojiljkovic’s laboratory is supported by the US National Scientific Foundation award # MCB 9728215 and US National Institutes of Health Public Service award #AI472870-01A1.
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Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores Cécile Wandersman* and Igor Stojiljkovic† The major mechanisms by which Gram-negative bacteria acquire heme from host heme-carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors. The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors. Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems. Addresses *Unité des Membranes Bactériennes, Institut Pasteur (CNRS URA 1300) 25 rue du Dr. Roux, 75724 Paris Cedex 15, France; e-mail: [email protected] † Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, USA; e-mail: [email protected] Current Opinion in Microbiology 2000, 3:215–220 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations Hb hemoglobin Hpt haptoglobin
Introduction Bacterial cells each contain 105 to 106 iron ions which are essential for many metabolic pathways. The low solubility of iron (III) salts at physiological pH in the presence of oxygen is a serious obstacle to microorganisms that require iron [1]. Microorganisms colonizing human hosts are confronted with another problem: 99.9% of total body iron (approx. 4 g) is intracellular and thus not readily available [2]. Furthermore, the extracellular iron found in human plasma and lymphatic fluids (a total of 3 mg) is tightly bound to transferrin. Small amounts of hemoglobin (Hb) (5–50 mg/L) are found in plasma as a result of spontaneous hemolysis. Free plasma Hb is bound by the circulating heterotetrameric glycoprotein, haptoglobin (Hpt), which delivers Hb dimers to the liver. Plasma albumin and hemopexin bind circulating heme and deliver it to the liver. Mucosal surfaces contain significant amounts of free heme and inorganic iron mostly complexed to lactoferrin and various metabolic byproducts [3]. Thus, human body fluids and surfaces contain sufficient amounts of inorganic, protein-bound and heme-associated iron to support bacterial growth. Therefore, for a bacterial pathogen to successfully colonize and establish in a mammalian host, it must be able to assimilate iron from (one or more of) these diverse sources of iron.
Iron assimilation Iron containing molecules are not transported across the outer membrane of Gram-negative bacteria by passive
diffusion: their extracellular concentrations are low and they are relatively large. Instead, these molecules are recognized and bound by specific receptors at the cell surface and actively transported against a concentration gradient into the bacterial periplasm. The iron capture systems can be classified in two groups: those that involve direct binding of iron or iron-containing proteins, and those that rely on the excretion of siderophores (low molecular weight iron ligands) and their receptors. Intact iron-loaded siderophores are taken up through the outer membrane, whereas iron-containing proteins are unloaded at the cell surface and only iron reaches the periplasm and is transported to the cytoplasm. Two siderophore-specific outer membrane receptors, FepA [4] and FhuA [5••], have been recently crystallised. Both are monomeric transmembrane β-barrels plugged on the periplasmic side by a amino-terminal cork-like domain of the protein. The activity of these receptors is dependent on a protein complex comprising two inner membrane proteins, ExbB and ExbD, and TonB, a proline-rich protein anchored to the cytoplasmic membrane via its amino terminus [6,7]. Experimental data have revealed direct interaction between TonB and the siderophore-specific outer membrane receptors [8]. Site-directed disulfide crosslinking demonstrated physical contact between a conserved amino-terminal region (the TonB-box) of the BtuB receptor and the TonB region around Gln160 [9]. Additional TonB and receptor domains must physically interact since a FhuA variant lacking its amino-terminal cork domain including the TonB-box was still able to take up siderophore in a TonB-dependent manner [10••]. The carboxy-terminal region of TonB is most probably involved in these interactions since carboxy-terminally truncated TonB constructs are not functional and are unable to interact physically with the FepA receptor [11].
Heme assimilation Just as in the case of intact iron-loaded siderophores, entire heme is taken up via outer membrane receptors and transported into the bacterial periplasm, whereas heme-containing proteins are unloaded at the cell surface and the heme is transported to the periplasm. Once in the periplasm, the heme is then transported by specific ABC protein-dependent periplasmic permeases through the inner membrane [12]. The fate of heme after entering the bacterial cytoplasm is not fully understood. However, at least one Gram-negative bacterium, Neisseriae, uses a heme oxygenase-like enzyme to acquire heme-iron [13]. Currently, twenty two different Gram-negative outer membrane proteins involved in the utilization of heme compounds are listed in the GeneBank data base. All
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share limited sequence identity (17% to 23%) with the outer membrane siderophore, transferrin and lactoferrin receptors, the amino terminus being the most conserved part of these proteins. Heme receptors have additional highly conserved domain (the FRAP/NPNL domain) [14••]. The overall structure of heme receptors is expected to be very similar to that of crystalized siderophore receptors. By analogy with iron uptake systems, we classify heme-uptake systems in two groups: first, those that involve direct binding of heme or heme-containing host proteins to specific outer membrane receptors, and second, those that rely on the secretion of hemophores (extracellular bacterial hemoproteins), which interact in the extracellular environment with the heme source and
present it to specific receptors (Figure 1). Here, we describe the different heme uptake systems, and focus on some important parallels and some significant differences with the systems for iron uptake.
Outer membrane receptors for heme and host hemoproteins
A first subgroup of outer membrane receptors includes the heme/Hb receptors of Yersinia, Escherichia coli and Shigella that share between 65% and 90% amino acid identity [15–18]. Studies on one member of this group, the Y. enterocolitica HemR, suggest that these receptors use many different heme containing-compounds as sources of iron [14••].
Figure 1
Gram-negative bacteria heme-uptake systems. (a) The HemR receptor of Y. enterocolitica. Heme/Hb binds the HemR receptor and heme is transported through the receptor channel in a TonB-dependent manner. See Figure 2 for more details. Other HemR-type receptors include ShuA from Shigella, ChuA from E. coli O157:H7, and HmuA from Y. pestis [15,16,18]. (b) The HmbR receptor of H. influenzae. The receptor is highly adapted to one or two heme–protein complexes. (c) The neisserial HpuAB receptor consists of two outer membrane proteins HpuA, a membrane lipoprotein, and HpuB, a Ton-dependent receptor. (d) Hemophores are secreted extracellularly where they bind
heme. The hemophere delivers the bound heme to a specific outer membrane receptor (HasR). The heme is transported into the cell by a TonB-dependent manner. The HasA family of hemophores are found in Y. pestis, P. aeruginosa, P. fluorescens and S. marcescens [35••]. The hemophore HxuA, which binds hemopexin, is found in H. influenzae [38]. The ABC protein is a membrane-associated ATPase belonging to the ATP-binding cassette protein superfamily [31]. The ABC protein is involved in heme transport from the periplasm to the cytoplasm. IM, inner membrane; OM, outer membrane; N, amino terminus; COOH, carboxyl terminus.
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Site-directed mutagenesis of the invariant histidine residue (His461) in the conserved FRAP/NPL domain in Y. enterocolitica HemR completely abolished the ability of the cell to feed on heme–protein complexes and severely impaired the use of free heme. Another highly conserved and functionally important histidine was identified at position 128 located in the putative cork-like domain of HemR. HemR receptor mutants in His128 were severely affected in the use of all heme sources, but still bind Hb–agarose to the outer membrane. These observations suggest that His128 and His461 participate in steps following the Hb–receptor contact. A hypothetical model (Figure 2) of HemR function postulates a TonB-dependent transfer of heme from the external His461 to the cork-positioned His128 such that heme is transported through the receptor channel [14••]. This transfer is accomplished by a change in heme-binding affinity of His461 and His128 that is generated by receptor–TonB interaction. As heme is buried within different hemoproteins with a very high affinity of association, it is unclear how it becomes available for interaction with HemR. The receptor may act as a surface chaperone and unfold the hemoproteins (see [19•]). However, HemR interacts with diverse hemoproteins that do not share significant similarity and no HemR–hemoprotein interactions have been shown biochemically, although they may be very weak and, therefore, not detectable by standard binding techniques. The second subgroup includes the heme receptors found in Vibrio, Neisseria and Haemophilus, including HutA, HmbR, HgbA, and Hpt–Hb receptors of H. influenzae [20–25]. These proteins are highly adapted to one or two heme–protein sources (Hb and/or Hpt–Hb complexes). They possess a high-affinity binding site for a particular heme–protein
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compound, and none of those tested allow growth on other heme–protein compounds. This suggests that these substrate-specific heme receptors directly recognize a protein carrier, whereas HemR and its homologs are able to recognize heme bound to diverse protein carriers.
The neisserial Hpt–Hb and Hb receptor, HpuAB, is a special case among the known heme receptors [26]. It consists of two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB). HpuB is TonB-dependent and similar to other single component TonB-dependent receptors (Figure 1). HpuA is an outer membrane lipoprotein. Both proteins are required for Hb, Hpt–Hb and apo-haptoglobin binding to whole cells. This bipartite hemoglobin receptor is very similar to the Neisseria transferrin (TbpAB) and lactoferrin (LbpAB) receptors [27]. Both components of the transferrin receptor are necessary for efficient transferrin binding; TbpA, the outer membrane TonB-dependent receptor, binds apo- and holo-transferrin similarily, whereas TbpB (the lipoprotein) discriminates between the loaded and unloaded substrate and binds ferrated transferrin with a 100 times higher affinity. The accessory lipoprotein, HpuA, may have a role very similar to that of TbpB. Unfortunately, it is not yet known whether HpuA and HpuB exhibit similar affinities for loaded and unloaded haptoglobin. Hemophore-dependent heme uptake
Hemophores (HasA) are small extracellular proteins produced by several Gram-negative bacteria [28–30]. They are secreted by ABC transporters, a process involving their carboxy-terminal secretion signal [31]. They form an independent family of heme-binding proteins without
Figure 2
A hypothetical mode of Y. enterocolitica HemR function. Heme binds external His461 and is transferred to the His128. This transfer is accomplished by a change in heme-binding affinity of His461 and His128 that is generated by a receptor–TonB interaction.
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homology to any other known proteins. The role of the hemophore is to bind heme and to deliver it to a specific outer membrane receptor, HasR. This receptor is essential for heme acquisition, whereas the hemophore is not, although it increases by a 100-fold the efficiency of the system for free or Hb-bound heme [32]. HasA is, however, essential for heme acquisition from other heme sources such as hemopexin and myoglobin (S Létoffé et al., unpublished data). By extracting heme from these sources, HasA supplies new heme substrate to HasR. Hence hemophores fulfill a function similar to that of siderophores: they are secreted to the extracellular medium and then return to the cell surface. They enhance the capacity of the system and broaden the spectrum of potential heme sources. It is also plausible but not demonstrated that they secure heme from other organisms. The Serratia marcescens HasA protein has been biochemically characterized. It exhibits a very high affinity for heme (Kd lower than 10–8 M), but does not undergo major structural changes upon heme binding [33]. The crystal structure of the heme–HasA complex has been resolved showing that the heme iron is highly exposed to the solvent and is bound to residues His32 and Tyr75 [34]. Both hemefree (apoprotein) and heme-loaded (holoprotein) HasA bind to HasR and they do so with similar apparent affinities (10–10 M), indicating direct protein–protein interactions between HasA and HasR [35••]. Apo-HasA bound to its outer membrane receptor, HasR, may either rapidly dissociate allowing a loaded molecule to bind or be able to bind heme present on holo-HasA. The HasA/HasR system is reminiscent of the Neisseria bipartite receptors HpuAB and TbpAB described above. HasA may have a function similar to that of the lipoproteins, HpuA and TbpB, enhancing the local heme concentration close to the receptor. Binding of the ligand-free form of the siderophore with an affinity similar to the loaded form has also been observed for a Pseudomonas aeruginosa siderophore pyoverdine [36•]. In this case, it is also not clear how the loaded form replaces the apo-siderophore and promotes iron transfer. The mechanism by which HasA extracts heme from hemoproteins is unknown. Clearly, heme transfer does not involve a stable complex between HasA and Hb [35••]. Heme capture from Hb by HasA might require either a transient protein complex or HasA to bind heme only when it spontaneously dissociates from hemoglobin. Another system involving hemophore has been described in H. influenzae. HxuA, which does not share sequence similarity with HasA, is secreted by a signal peptide-dependent pathway involving one outer membrane helper protein. HxuA is able to acquire heme from hemopexin and to return it to a specific receptor in the outer membrane [37,38].
The function of TonB in heme acquisition Various studies show the presence of highly conserved TonB proteins in all Gram-negative bacteria and suggest
the importance of TonB in heme acquisition processes. However, the available data concerning the role of TonB in heme uptake do not elucidate its molecular mechanism, despite the X-ray crystallography data obtained for two outer membrane siderophore receptors. TonB may also be involved in steps other than heme transport itself, in particular the binding of heme, hemoprotein and hemophore to their respective outer membrane receptors, and heme striping from heme carriers. In all studies done so far binding of hemoproteins did not depend on the presence of TonB ([35••]; M Baer et al., unpublished data). However, the kinetics of binding of hemoproteins to the receptors have not been studied and thus there is a possibility that parameters of binding, such as affinity and off/on rates, might be quite different between TonB+ and TonB– cells. Several lines of evidence indicate that the subsequent step of heme transfer from the heme carrier to the receptor might be TonB dependent. Data for diverse Hb receptors show that the unloading occurs only in TonB+ strains [39,40]. However, these experiments did not clearly dissociate the unloading step from the uptake step, which is TonB dependent. Heme uptake involves the receptors being unloaded of heme and thereby uptake generates new available sites. Previously, it was thought that TonB was unique and was shared by most of the iron uptake systems in each species. However, several recent studies have indicated the existence of TonB proteins that specialise in heme and hemoprotein uptake. In V. cholerae, two sets of tonB, exbB, and exbD genes have been identified [41••]. They have at least partially redundant functions since inactivation of each set did not result in a defect in iron or heme uptake. The tonB1 locus was shown to be part of the hut operon dedicated to heme uptake. DNA sequence data show the presence of such analogs also in Pseudomonas aeruginosa and S. marcescens ([42]; JM Ghigo et al., unpublished data). Redundancy of TonBs in these species is most likely to be evidence of a distant horizontal spread of heme-uptake operons between different microorganisms. Since receptor–TonB interactions are to some extent species-specific, a newly acquired heme uptake system can only be fully exploited if the heme receptor is accompanied by its cognate TonB protein. Further, an increase in the amount of TonB in the cell may make the transport of TonB-dependent substrates more efficient since different receptors normally compete for limiting amounts of TonB [43].
Concluding remarks Notable progress has been made during the past decade in the identification of bacterial surface receptors for hemecontaining compounds. These studies reveal that heme receptors, unlike siderophore receptors, have at least two distinct structural designs. The large number of different heme-containing compounds present in the biosphere was probably the major driving force behind the evolution of different types of heme receptors. Partial redundancy of
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heme systems in Gram-negative bacteria may be a consequence of the diversity of heme sources in the environment and an adaptation to rapidly changing amounts and varieties of hemoproteins encountered during various stages of infection. This redundancy together with the phase variation of receptor expression must also be a successful protection strategy against immune surveillance in the host [44–47]. Indeed, previous work has established that heme receptors are expressed in vivo during infection, and that the ability to use heme and Hb contributes to the colonization potential and virulence of pathogenic bacteria [21,48–51]. All heme receptors share significant amino acid sequence similarity with siderophore receptors indicating a common mechanism. However, heme receptors are confronted with more complicated task than siderophore receptors since they deal with heme tightly bound to diverse protein carriers. Currently, it is not clear how receptors extract heme from heme carrier proteins and hemophores. It is unlikely that recently discovered proteases, able to cleave Hb, are the answer to this problem [52,53]. Therefore, to unravel the mechanism of heme-receptor function will probably require deciphering the nature of interaction between receptors and heme-carrier proteins and defining the role of the TonB protein in these interactions.
Acknowledgements We thank Sylvie Létoffé and Jean Marc Ghigo for helpful discussions. The work in Igor Skojiljkovic’s laboratory is supported by the US National Scientific Foundation award # MCB 9728215 and US National Institutes of Health Public Service award #AI472870-01A1.
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